JP2019104744A - Use of aromatic-cationic peptides to treat cholesterol-induced mitochondrial dysfunction - Google Patents

Abstract

To provide aromatic-cationic peptide compositions and methods of using the same.SOLUTION: The present disclosure provides aromatic-cationic peptide compositions and methods of using the same. The methods comprise use of the peptides in inhibiting cholesterol-induced cytochrome c peroxidase activity, promoting reduction of cytochrome c, and inhibiting cardiolipin peroxidation, prevent or ameliorate cholesterol-induced mitochondrial dysfunction.SELECTED DRAWING: Figure 1

Description

This application claims the benefit of and priority to US Application No. 61 / 883,513, filed September 27, 2013, which is incorporated herein by reference in its entirety.

  The present technology relates generally to aromatic-cationic peptide compositions and methods of use in the treatment, prevention or amelioration of cholesterol-induced mitochondrial dysfunction.

  The following explanations are provided to assist the reader's understanding. Neither the information provided nor the references cited are acknowledged as prior art.

  High cholesterol is responsible for most deaths worldwide. Recently, mitochondrial dysfunction has been associated with a number of different diseases across diseases such as cancer, Parkinson's disease, diabetes and Alzheimer's disease (Kiebish et al., 2008, Montero et al., 2010, Paradies et al., 2009). Mitochondrial dysfunction and high cholesterol have been shown to have various adverse effects on cells.

  In one aspect, the subject technology suffers from a disease characterized by cholesterol induced mitochondrial dysfunction, comprising administering to the subject a therapeutically effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof. Provide a way to treat. In some embodiments, the salt is acetate, tartrate, or trifluoroacetate.

In some embodiments of the method, the aromatic cationic peptides, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys -NH ₂ (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31). In certain embodiments of the present method, aromatic cationic peptides, D-Arg-2 ', including 6'Dmt-Lys-Phe-NH ₂ a (SS-31).

  In some embodiments of the method, the disease is Niemann-Pick disease.

  In some embodiments of the method, the treatment comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, wherein the symptoms of Niemann-Pick disease include difficulty moving the limbs (dystonia), spleen and Liver hypertrophy (hepatosplenomegaly), dementia, indistinct and irregular speech (arthria), dysphoria (dysphagia), sudden loss of muscle tone that can lead to falls (acute seizures), tremor War, difficulty moving the eye up and down (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlarged marrow cavity, thinning of cortical bone, hip bone One or more symptoms selected from the group consisting of strain, seizures, cherry red spots in the eye, and sleep related disorders.

  In some embodiments of the method, the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.

  In another aspect, the present technology comprises administering an effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to a subject, in a subject suffering from a disease characterized by cholesterol-induced mitochondrial dysfunction Provided is a method for reducing cardiolipin oxidation. In some embodiments, the salt is acetate, tartrate, or trifluoroacetate.

In some embodiments of the method, the aromatic cationic peptides, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys -NH ₂ (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31). In certain embodiments of the present method, aromatic cationic peptide comprises D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31).

  In some embodiments of the method, the disease is Niemann-Pick disease.

  In some embodiments of the method, the treatment comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, wherein the symptoms of Niemann-Pick disease include difficulty moving the limbs (dystonia), spleen and Liver hypertrophy (hepatosplenomegaly), dementia, indistinct and irregular speech (arthria), dysphoria (dysphagia), sudden loss of muscle tone that can lead to falls (acute seizures), tremor War, difficulty moving the eye up and down (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlarged marrow cavity, thinning of cortical bone, hip bone One or more symptoms selected from the group consisting of strain, seizures, cherry red spots in the eye, and sleep related disorders.

  In some embodiments of the method, the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.

  In another aspect, the present technology comprises administering an effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to a subject, in a subject suffering from a disease characterized by cholesterol-induced mitochondrial dysfunction Provided are methods for reducing cholesterol-induced cytochrome c peroxidase activity.

In some embodiments of the method, the aromatic cationic peptides, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys -NH ₂ (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31).

  In some embodiments of the method, the salt is acetate, tartrate, or trifluoroacetate.

  In some embodiments of the method, the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.

  In some embodiments of the method, the disease is Niemann-Pick disease.

  In some embodiments of the method, the treatment comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, difficulty moving the limbs (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly Disease, dementia, indistinct and irregular speech (arthria), dysphoria (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, moving eyes up and down Difficulty (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of cortical bone, distortion of hip bone, seizure, in the eye Cherry red spots, as well as symptoms of sleep related disorders.

  In another aspect, the present technology comprises administering an effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to a subject, in a subject suffering from a disease characterized by cholesterol-induced mitochondrial dysfunction Provided is a method for protecting cytochrome c from cholesterol induced structural damage.

In some embodiments of the method, the aromatic cationic peptides, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys -NH ₂ (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31).

  In some embodiments of the method, the salt is acetate, tartrate, or trifluoroacetate.

  In some embodiments of the method, the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.

  In some embodiments of the method, the disease is Niemann-Pick disease.

  In some embodiments of the method, the treatment comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, difficulty moving the limbs (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly Disease, dementia, indistinct and irregular speech (arthria), dysphoria (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, moving eyes up and down Difficulty (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of cortical bone, distortion of hip bone, seizure, in the eye Cherry red spots, as well as symptoms of sleep related disorders.

FIG. 6 shows that cholesterol increases cytochrome c peroxidase activity in a dose-dependent manner. An increase in cytochrome c peroxidase activity is an increase in the concentration of cholesterol in the form of free cholesterol (represented by black circles) or chol-POPC (represented by gray squares) relative to 2 μM cytochrome c (0-300 μM). Cholesterol). Cytochrome c peroxidase activity was measured using the fluorescence of 50 μM Amplex Red reagent in the presence of 10 μM H ₂ O ₂ . D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31) peptide (filled expressed in bars) and Phe-D-Arg-Phe- Lys-NH 2 peptide (SS-20) (Represented by a hatched bar) is shown to inhibit cholesterol-induced cytochrome c peroxidase activity in a dose dependent manner. Various D-Arg-2 concentration 'range of 0μM~300μM, 6'-Dmt-Lys- Phe-NH 2 (SS-31) and Phe-D-Arg-Phe- Lys-NH 2 (SS-20 The addition of) significantly reduced the effect of 300 μM cholesterol on the induction of peroxidase activity of 2 μM cytochrome c. Cytochrome c peroxidase activity was measured using the fluorescence of 50 μM Amplex Red reagent in the presence of 10 μM H ₂ O ₂ . Figure 3A-B is treatment with D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31) and Phe-D-Arg-Phe- Lys-NH 2 (SS-20) Is shown to alleviate the effect of cholesterol on cytochrome c reduction. FIG. 3A shows that the addition of increasing amounts of cholesterol exhibits a reduction in the reducing ability of cytochrome c as compared to the reduction of cytochrome c without cholesterol. Chol-POPC liposomes were added to 20 μM cytochrome c in 20 mM Hepes buffer pH 7.4 and 50 μM vitamin C. Figure 3A-B is treatment with D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31) and Phe-D-Arg-Phe- Lys-NH 2 (SS-20) Is shown to alleviate the effect of cholesterol on cytochrome c reduction. FIG. 3B shows 100 μM chol-POPC liposomes with 20 μM cytochrome c (in 20 mM Hepes buffer pH 7.4 and 50 μM vitamin C) and different concentrations of D-Arg-2 ranging from 0 to 300 μM. ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) peptide (represented by solid bars) and Phe-D-Arg-Phe-Lys-NH ₂ (SS-20) peptide (with hatched bars) The effect of the concentration pretreated with one of the The results showed that as the peptide concentration was increased, the reducing ability of cytochrome c was significantly increased. D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31) and Phe-D-Arg-Phe- Lys-NH 2 (SS-20) is the reducing ability of cytochrome c, It was returned to its initial state prior to treatment with 100 μM chol-POPC liposomes. 6 is a CD spectrum showing that the addition of peptide D-Arg-2 ′, 6′-Dmt-Lys-Phe-NH ₂ (SS-31) inhibits cholesterol-induced structural changes of cytochrome c. The addition of peptide restored some of the peaks lost due to cholesterol induced cytochrome c damage. The addition of 40 μM chol-POPC significantly changes the CD spectrum, and 100 μM D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) gives the spectrum almost at its initial condition. I recovered. 5A-B show that D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) peptide reduces cholesterol-induced changes to the absorption spectrum of cytochrome c. is there. FIG. 5A shows a scan of the absorption spectrum of cytochrome c incubated with cytochrome c and chol-POPC liposomes. These results show significant structural changes of cytochrome c protein upon exposure to chol-POPC liposomes. 5A-B show that D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) peptide reduces cholesterol-induced changes to the absorption spectrum of cytochrome c. is there. Figure 5B shows that D-Arg-2 ', the addition of 6'-Dmt-Lys-Phe- NH 2 (SS-31), reduces the influence of chol-POPC liposomes against cytochrome c structure. D-Arg-2 ', is a diagram showing an emission spectrum of 6'-Dmt-Lys-Ald- NH 2 ([ald] SS-31). These results indicate that the peptide blocks the formation of cholesterol-cytochrome c complex.

  It is understood that certain aspects, configurations, embodiments, variations, and features of the present technology are described below at various levels of detail to provide a substantial understanding of the present technology. Definitions of certain terms that may be used herein are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

  In practicing the present disclosure, many conventional techniques of cell biology, molecular biology, protein biochemistry, immunology, and bacteriology are used. These techniques are well known in the art and described in Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997), Sambrook et al. , Molecular Cloning: A Laboratory Manual, Second Ed. It is provided in a number of available publications, including (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

  As used in this specification and the appended claims, the singular forms "a", "an", and "the" are used, unless the context clearly dictates otherwise. , Including multiple referents. For example, reference to "a cell" includes a combination of two or more cells and the like.

  As used herein, "administration" of a drug, drug, or peptide to a subject includes any route of introduction or delivery of a compound to the subject to perform its intended function. Administration can be oral, nasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), topical, on the skin, rectal, intradermal, transdermal, inhalation, intraarterial, intracerebral, interosseous, spinal It can be performed by any suitable route, including intraluminal, iontophoresis, transocular, vaginal, and the like. Administration includes self-administration and administration by others.

  As used herein, the term "amino acid" includes naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. An amino acid analog is a compound having the same basic chemical structure as a naturally occurring amino acid, ie, hydrogen, a carboxyl group, an amino group, and an α-carbon linked to an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium Point to. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by any of their well-known three letter symbols, or one letter symbols, recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

  As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect. In the context of therapeutic or prophylactic use, the amount of composition administered to a subject will depend on the type and severity of the disease, and individual characteristics such as general health, age, sex, weight, and drug resistance. become. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions may also be administered in combination with one or more additional therapeutic compounds.

  The "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminating polypeptide from the cell or tissue source from which the agent is derived, or is chemically synthesized. When substantially free of chemical precursors or other chemicals. For example, the isolated aromatic-cationic peptide or the isolated cytochrome c protein does not contain substances which interfere with the diagnostic or therapeutic use of the drug or which interfere with the conductivity or electrical properties of the peptide . Such interferents can include enzymes, hormones, and other proteinaceous and nonproteinaceous solutes.

  As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are two or more linked together by peptide bonds or modified peptide bonds. By amino acid is meant a polymer that contains the peptide isostere. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and long chains, commonly referred to as proteins. The polypeptide may comprise amino acids other than the 20 genetically encoded amino acids. Polypeptides include amino acid sequences that have been modified by either natural processes such as posttranslational processing or chemical modification techniques that are well known in the art.

  As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

  As used herein, the term "sequential" therapeutic use refers to administering at least two active ingredients at different times, and the routes of administration may be the same or different. More specifically, sequential use refers to complete administration of one of the active ingredients prior to commencing administration of the other active ingredient (s). Thus, one of the active ingredients can be administered over several minutes, hours or days before administering the other active ingredient (s). There is no concurrent treatment in this case.

  As used herein, the term "simultaneous" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by the same route.

  As used herein, the terms "subject", "indivisual" or "patient" may be an individual organism, a vertebrate, a mammal, or a human it can.

  As used herein, a "synergistic therapeutic effect" is an additive therapeutic effect that is produced by the combination of at least two therapeutic agents and exceeds that which would otherwise result from the single administration of at least two therapeutic agents. (Greater-than-additive therapeutic effect). Thus, the use of low doses of one or more of at least two therapeutic agents for the treatment of a disease or disorder may result in increased therapeutic efficacy and reduced side effects.

  As used herein, a "therapeutically effective amount" of a compound refers to a compound level at which the physiological effects of the disease or disorder are at least ameliorated. A therapeutically effective amount can be given in one or more administrations. The amount of compound that makes up a therapeutically effective amount will vary depending on the compound, the disease and its severity, and the general health, age, sex, weight, and drug resistance of the subject being treated. It can be routinely determined by the vendor.

  As used herein, the terms "treating" or "treatment" or "alleviation" refer to a disease or disorder described herein in a subject such as a human. Covering the treatment, (i) inhibition of the disease or disorder, ie cessation of its onset, (ii) alleviation of the disease or disorder, ie induction of regression of the disease, (iii) slowing of the progression of the disease, and / or / or (Iv) involves the inhibition, alleviation, or slowing of the progression of one or more symptoms of the disease or disorder.

  As used herein, "prevention" or "preventing" of a disease or condition reduces the incidence of the disease or condition in the treated sample relative to untreated control samples. Refers to a compound that delays the onset of one or more symptoms of a disease or condition as compared to a control sample that is allowed to or remains untreated.

  Also, as the various forms of treatment or prevention of a medical condition as described means "substantial" including complete treatment or prevention as well as less than complete treatment or prevention. It is also to be understood that intended and biologically relevant or medically relevant results are achieved.

Aromatic Cationic Peptides The present technology relates to the use of aromatic cationic peptides. In some embodiments, the peptides are useful in aspects relating to treating or ameliorating a condition, condition or disease characterized by cholesterol-induced mitochondrial dysfunction.

  Aromatic cationic peptides are water soluble and highly polar. Notwithstanding these properties, the peptide can readily penetrate cell membranes. Aromatic cationic peptides typically comprise a minimum of 3 amino acids or a minimum of 4 amino acids covalently linked by peptide bonds. The maximum number of amino acids present in the aromatic-cationic peptide is about 20 amino acids covalently linked by peptide bonds. Preferably, the maximum number of amino acids is about 12, about 9, or about 6.

  The amino acid of the aromatic cationic peptide can be any amino acid. As used herein, the term "amino acid" is used to refer to any organic molecule comprising at least one amino group and at least one carboxyl group. Typically, at least one amino group is alpha to the carboxyl group. The amino acids may be naturally occurring. As natural amino acids, for example, the 20 most common levo-rotatory (L) amino acids normally found in mammalian proteins, ie, alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid ( Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine ( Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, (Trp), tyrosine (Tyr), and valine (Val). Other naturally occurring amino acids include, for example, amino acids synthesized during metabolic processes not associated with protein synthesis. For example, the amino acids ornithine and citrulline are synthesized in mammalian metabolism during the production of urea. Another example of a naturally occurring amino acid is hydroxyproline (Hyp).

  The peptide optionally comprises one or more non-naturally occurring amino acids. Optimally, the peptide does not have naturally occurring amino acids. The non-naturally occurring amino acid may be levorotatory (L-), dextrorotatory (D-), or a mixture thereof. Non-naturally occurring amino acids are amino acids that are typically not synthesized in normal metabolic processes in living organisms and are not naturally present in proteins. In addition, non-naturally occurring amino acids are also preferably not recognized by common proteases. Non-naturally occurring amino acids can be present at any position in the peptide. For example, the non-naturally occurring amino acid can be present at the N-terminus, C-terminus, or any position between the N-terminus and C-terminus.

  Non-naturally occurring amino acids can include, for example, alkyl, aryl or alkylaryl groups not found in naturally occurring amino acids. Some examples of non-naturally occurring alkyl amino acids include alpha-aminobutyric acid, beta-aminobutyric acid, gamma-aminobutyric acid, delta-aminovaleric acid, and epsilon-aminocaproic acid. Some examples of non-naturally occurring aryl amino acids include ortho, meta and paraaminobenzoic acid. Some examples of non-naturally occurring alkylaryl amino acids include ortho, meta, and paraaminophenylacetic acid, and γ-phenyl-β-aminobutyric acid. Non-naturally occurring amino acids include derivatives of naturally occurring amino acids. Derivatives of naturally occurring amino acids may include, for example, the addition of one or more chemical groups to naturally occurring amino acids.

For example, the one or more chemical groups may be one or more of the 2 ', 3', 4 ', 5' or 6 'position of the aromatic ring of the phenylalanine or tyrosine residue or the benzo ring of the tryptophan residue. It can be attached to one or more of the 4 ', 5', 6 'or 7' positions. The group may be any chemical group that can be attached to the aromatic ring. Some examples of such groups are branched or unbranched C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyloxy such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl or t-butyl (i.e., alkoxy), amino, C ₁ -C ₄ alkylamino, and C ₁ -C ₄ dialkylamino (e.g., methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro, chloro, bromo or iodo, Can be mentioned. Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include norvaline (Nva) and norleucine (Nle).

Another example of amino acid modification in peptides is derivatization of the carboxyl group of the aspartic acid or glutamic acid residue of the peptide. An example of derivatization is amidation with ammonia or primary or secondary amines such as methylamine, ethylamine, dimethylamine or diethylamine. Another example of derivatization includes, for example, esterification with methyl or ethyl alcohol. Another such modification includes derivatization of the amino group of lysine, arginine or histidine residues. For example, such amino groups can be acrylated. Some suitable acyl groups include, for example, benzoyl or alkanoyl groups comprising any of the aforementioned C ₁ -C ₄ alkyl groups, such as acetyl or propionyl groups.

  Non-naturally occurring amino acids are preferably resistant or insensitive to common proteases. Examples of non-naturally occurring amino acids that are resistant or insensitive to proteases include the above-mentioned naturally occurring L-amino acids and the non-naturally occurring L- and / or D-amino acids of the dextrorotatory (D- ) Form is mentioned. D-amino acids are usually found in certain peptide antibiotics that do not occur in proteins but are synthesized by means other than the cell's normal ribosomal protein synthesis machinery. As used herein, D-amino acids are considered to be non-naturally occurring amino acids.

  In order to minimize protease sensitivity, peptides are less than 5, less than 4, less than 3 recognized by common proteases, regardless of whether the amino acids are naturally occurring or non-naturally occurring. Or should have less than 2 contiguous L-amino acids. In one embodiment, the peptide has only D-amino acids and no L-amino acids. When the peptide comprises a protease sensitive sequence of amino acids, it is preferred that at least one of the amino acids be non-naturally occurring D-amino acid to confer protease resistance. Examples of protease sensitive sequences include two or more contiguous basic amino acids that are easily cleaved by common proteases, such as endopeptidase and trypsin. Examples of basic amino acids include arginine, lysine and histidine.

The aromatic-cationic peptide should have a minimum number of net positive charges at physiological pH as compared to the total number of amino acid residues in the peptide. The minimum number of net positive charges at physiological pH is referred to below as (p _m ). The total number of amino acid residues in the peptide is referred to below as (r). The minimum number of net positive charges discussed below are all at physiological pH. The term "physiological pH" as used herein refers to the normal pH in cells of tissues and organs of the mammalian body. For example, human physiological pH is usually about 7.4, but normal physiological pH in mammals can be any pH in the range of about 7.0 to about 7.8.

  "Net charge" as used herein refers to the difference between the number of positive charges carried by the amino acids present in the peptide and the number of negative charges. It is understood herein that net charge is measured at physiological pH. Naturally occurring amino acids positively charged at physiological pH include L-lysine, L-arginine, and L-histidine. Natural amino acids negatively charged at physiological pH include L-aspartic acid and L-glutamic acid.

  Typically, the peptide has a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. These charges are mutually offset at physiological pH. As an example of calculating the net charge, the Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg peptide consists of one negatively charged amino acid (ie Glu) and four positively charged amino acids (Ie 2 Arg residues, 1 Lys and 1 His). Thus, the above peptide has 3 net positive charges.

In one embodiment, the aromatic cationic peptides, 3p _m is the maximum number of less than or equal to r + 1, the minimum number of net positive charges at physiological pH (p _m) and the total number of amino acid residues (r) and the Have a relationship. In this embodiment, the relationship between the minimum number of net positive charge and (p _m) and the total number of amino acid residues (r) is as follows.

In another embodiment, the aromatic cationic peptides, 2p _m is the maximum number of less than or equal to r + 1, have the relationship of the minimum number of net positive charge and (p _m) and the total number of amino acid residues (r). In this embodiment, the relationship between the minimum number of net positive charge and (p _m) and the total number of amino acid residues (r) is as follows.

In one embodiment, the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r) are equal. In another embodiment, the peptide has 3 or 4 amino acid residues and a minimum of 1 net positive charge, preferably a minimum of 2 net positive charges, more preferably a minimum of 3 Have a net positive charge of

It is also important that the aromatic-cationic peptide have a minimal number of aromatic groups as compared to the total number of net positive charges ( _pt ). The minimum number of aromatic groups is referred to below as (a). Natural amino acids having an aromatic group include amino acids such as histidine, tryptophan, tyrosine and phenylalanine. For example, Lys-Gln-Tyr-D-Arg-Phe-Trp hexapeptide has two net positive charges (contributed by lysine and arginine residues), and three aromatic groups (tyrosine residues , Phenylalanine residue, and tryptophan residue).

Aromatic cationic peptides are also physiologically compatible with the minimum number of aromatic groups (a), where 3a is the largest number less than p _t +1, except that a can also be 1 when p _t is 1. It has a relationship with the total number of net positive charges ( _pt ) at pH. In this embodiment, the relationship between the minimum number of aromatic groups (a) and the total number of net positive charges ( _pt ) is as follows.

In another embodiment, the aromatic cationic peptides, 2a is the largest number equal to or less than p _t +1, has a relationship of the minimum number of aromatic groups (a) and the total number of net positive charge and (p _t) . In this embodiment, the relationship between the minimum number of aromatic amino acid residues (a) and the total number of net positive charges ( _pt ) is as follows.

In another embodiment, the number of aromatic groups (a) and the total number of net positive charges ( _pt ) are equal.

The carboxyl group, in particular the terminal carboxyl group of the C-terminal amino acid, is suitably amidated, for example with ammonia, to form a C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid can be amidated with any primary or secondary amine. The primary or secondary amine may, for example, be an alkyl, in particular a branched or unbranched C ₁ -C ₄ alkyl or arylamine. Thus, the amino acid at the C-terminus of the peptide is amide, N-methylamide, N-ethylamide, N, N-dimethylamide, N, N-diethylamide, N-methyl-N-ethylamide, N-phenylamide, N-phenylamide It can be converted to -N-ethylamide group. Free carboxylate groups of asparagine, glutamine, aspartic acid, and glutamic acid residues that do not occur at the C-terminus of the aromatic cationic peptide can also be amidated if they occur in the peptide. Amidation at these internal positions may be performed with either ammonia or primary or secondary amines as described above.

  In one embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and at least one aromatic amino acid. In certain embodiments, the aromatic-cationic peptide is a tripeptide having two net positive charges and two aromatic amino acids.

In another aspect, the present technology provides an aromatic cationic peptide, or a pharmaceutically acceptable salt thereof such as acetate, tartrate, or trifluoroacetate. In some embodiments, the peptide is
1. At least one net positive charge,
2. At least 3 amino acids,
3. Up to about 20 amino acids,
4. The relationship between the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r), the maximum number of which is 4.3 p _m is r + 1 or less, and except that a can be a p _t even 1 when it is 1, 2a is the largest number equal to or less than p _t +1, the total minimum number (a) and net positive charge of the aromatic group (p _t Including the relationship with).

In some embodiments, the peptide has the amino acid sequence Tyr-D-Arg-Phe-Lys-NH ₂ (SS-01), 2 ', 6'-Dmt-D-Arg-Phe-Lys-NH ₂ (SS -02), including Phe-D-Arg-Phe- Lys-NH 2 (SS-20), or D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31). In some embodiments, the peptide is
D-Arg-Dmt-Lys-Trp-NH ₂ ,
D-Arg-Trp-Lys-Trp-NH ₂ ,
D-Arg-2 ', 6' -Dmt-Lys-Phe-Met-NH 2,
HD-Arg-Dmt-Lys (N ^α Me) -Phe-NH ₂ ,
H-D-Arg-Dmt- Lys-Phe (NMe) -NH 2,
HD-Arg-Dmt-Lys (N ^α Me) -Phe (NMe) -NH ₂ ,
^{H-D-Arg (N α} Me) -Dmt (NMe) -Lys (N α Me) -Phe (NMe) -NH 2,
D-Arg-Dmt-Lys-Phe-Lys-Trp-NH ₂ ,
D-Arg-Dmt-Lys- Dmt-Lys-Trp-NH 2,
D-Arg-Dmt-Lys-Phe-Lys-Met-NH ₂ ,
D-Arg-Dmt-Lys-Dmt-Lys-Met-NH ₂ ,
H-D-Arg-Dmt- Lys-Phe-Sar-Gly-Cys-NH 2,
HD-Arg-Arg [CH ₂ -NH] Dmt-Lys-Phe-NH ₂ ,
HD-Arg-Dmt-mt [CH ₂ -NH] Lys-Phe-NH ₂ ,
H-D-Arg-Dmt-LysΨ [CH ₂ -NH] Phe-NH ₂ ,
HD-Arg-Dmt-mt [CH ₂ -NH] Lys-Ψ [CH ₂ -NH] Phe-NH ₂ ,
Lys-D-Arg-Tyr- NH 2,
Tyr-D-Arg-Phe-Lys-NH ₂ ,
2 ', 6'-Dmt-D -Arg-Phe-Lys-NH 2,
Phe-D-Arg-Phe- Lys-NH 2,
Phe-D-Arg-Dmt- Lys-NH 2,
D-Arg-2'6'Dmt-Lys- Phe-NH 2,
H-Phe-D-Arg- Phe-Lys-Cys-NH 2,
Lys-D-Arg-Tyr- NH 2,
D-Tyr-Trp-Lys-NH ₂ ,
Trp-D-Lys-Tyr-Arg-NH ₂ ,
Tyr-His-D-Gly-Met,
Tyr-D-Arg-Phe-Lys-Glu-NH ₂ ,
Met-Tyr-D-Lys-Phe-Arg,
D-His-Glu-Lys-Tyr-D-Phe-Arg,
Lys-D-Gln-Tyr- Arg-D-Phe-Trp-NH 2,
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH ₂ ,
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH ₂ , Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,
Lys-Trp-D-Tyr- Arg-Asn-Phe-Tyr-D-His-NH 2, Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys, Asp-D-Trp -Lys-Tyr-D-His- Phe-Arg-D-Gly-Lys-NH 2,
D-His-Lys-Tyr- D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH 2,
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,
Phe-Phe-D-Tyr- Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH 2,
Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,
Glu-Arg-D-Lys- Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH 2,
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-P-Phe-Tyr-D-Arg-Gly,
D-Glu-Asp-Lys- D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH 2,
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-P-Ly-Phe,
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH ₂ , Gly-Ala-Lys -Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp;
Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys- NH ₂ ,
(Atn) Dap is β- anthraniloyl -L-alpha, a β- diaminopropionic acid, Dmt-D-Arg-Phe- (atn) Dap-NH 2,
Dmt-D-Arg-Ald-Lys-NH ₂ , wherein Ald is β- (6'-dimethylamino-2'-naphthoyl) alanine,
Dmt-D-Arg-Phe-Lys-Ald-NH ₂ , wherein Ald is β- (6'-dimethylamino-2'-naphthoyl) alanine;
(Dns) Dap is β- dansyl -L-alpha, a β- diaminopropionic acid, Dmt-D-Arg-Phe- (dns) Dap-NH 2,
It includes one or more of D-Arg-Tyr-Lys-Phe-NH ₂ and D-Arg-Tyr-Lys-Phe-NH ₂ .

  In some embodiments, “Dmt” refers to 2 ′, 6′-dimethyltyrosine (2 ′, 6′-Dmt) or 3 ′, 5′-dimethyltyrosine (3′5′Dmt).

In some embodiments, the peptide is defined by Formula I:
In the formula, R ¹ and R ² are each independently
(I) hydrogen,
(Ii) straight or branched C ₁ -C ₆ alkyl,
Is selected from
R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each independently
(I) hydrogen,
(Ii) linear or branched C ₁ -C ₆ alkyl,
(Iii) C ₁ -C ₆ alkoxy,
(Iv) amino,
(V) C ₁ -C ₄ alkylamino,
(Vi) C ₁ -C ₄ dialkylamino,
(Vii) nitro,
(Viii) hydroxyl,
(Ix) selected from halogens including chloro, fluoro, bromo and iodo;
n is an integer of 1 to 5;

In some embodiments, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are all hydrogen, n is four. In another embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹¹ are all hydrogen and R ⁸ and R ¹² are Methyl, R ¹⁰ is hydroxyl and n is 4;

In some embodiments, the peptide is defined by Formula II:
In the formula, R ¹ and R ² are each independently
(I) hydrogen,
(Ii) straight or branched C ₁ -C ₆ alkyl,
Is selected from
R ³ and R ⁴ are each independently
(I) hydrogen,
(Ii) linear or branched C ₁ -C ₆ alkyl,
(Iii) C ₁ -C ₆ alkoxy,
(Iv) amino,
(V) C ₁ -C ₄ alkylamino,
(Vi) C ₁ -C ₄ dialkylamino,
(Vii) nitro,
(Viii) hydroxyl,
(Ix) selected from halogens including chloro, fluoro, bromo and iodo;
R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are each independently
(I) hydrogen,
(Ii) linear or branched C ₁ -C ₆ alkyl,
(Iii) C ₁ -C ₆ alkoxy,
(Iv) amino,
(V) C ₁ -C ₄ alkylamino,
(Vi) C ₁ -C ₄ dialkylamino,
(Vii) nitro,
(Viii) hydroxyl,
(Ix) selected from halogens including chloro, fluoro, bromo and iodo;
n is an integer of 1 to 5;

In some embodiments, the peptide is defined by the following formula:
The 2 ', represented as 6'-Dmt-D-Arg- Phe- (dns) Dap-NH 2, wherein, (dns) Dap is, beta-dansyl -L-alpha, beta-diaminopropionic acid (SS -17).

In some embodiments, the peptide is defined by the following formula:
It is also expressed as 2 ', 6'-Dmt-D-Arg-Phe- (atn) Dap-NH ₂ , wherein (atn) Dap is β-anthranyloyl-L-α, β-diaminopropionic acid (SS -19). SS-19 is also referred to as [atn] SS-02.

In certain embodiments, R ¹ and R ² are hydrogen, R ³ and R ⁴ are methyl, R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are all hydrogen and n is 4

In one embodiment, the aromatic-cationic peptide has a core structural motif of alternating aromatic and cationic amino acids. For example, the peptide may be a tetrapeptide as defined by any of Formulas III-VI shown below:
Aromatic-cationic-aromatic-cationic (formula III)
Cationic-Aromatic-Cationic-Aromatic (Formula IV)
Aromatic-aromatic-cationic-cationic (formula V)
Cationic-cationic-aromatic-aromatic (formula VI)
In the formula, aromatic is a residue selected from the group consisting of Phe (F), Tyr (Y), Trp (W), and cyclohexylalanine (Cha), and cationic is , Lys (K), norleucine (Nle), and 2-amino-heptanoic acid (Ahe).

  In some embodiments, the aromatic-cationic peptide described herein comprises all levorotatory (L) amino acids.

In one embodiment, the aromatic-cationic peptide is
1. At least one net positive charge,
2. At least 3 amino acids,
3. Up to about 20 amino acids,
4. The relationship between the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r), the maximum number of which is 4.3 p _m is r + 1 or less, and except that a can be a p _t even 1 when it is 1, 2a is the largest number equal to or less than p _t +1, the total minimum number (a) and net positive charge of the aromatic group (p _t Have a relationship with

In another embodiment, the present technology
A condition that reduces the number of mitochondria undergoing mitochondrial permeability transition (MPT), or prevents mitochondrial permeability transition in excised organs of the mammal, or is characterized by cholesterol-induced mitochondrial dysfunction, Provided are methods for treating a condition or disease. The method comprises administering an effective amount of an aromatic cationic peptide to the excised organ, the effective amount of the aromatic cationic peptide comprising
At least one net positive charge,
At least 3 amino acids,
Up to about 20 amino acids,
The relationship between the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r), where 3 p _m is the largest number less than r + 1, and p _t can also be 1 if a is 1 Except that 2a is the maximum number less than p _t +1, it has a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p _t ).

In another embodiment, the present technology reduces the number of mitochondria undergoing mitochondrial permeability transition (MPT) or prevents mitochondrial permeability transition in a mammal in need thereof Or provide a method for treating or ameliorating a condition, condition or disease characterized by cholesterol induced mitochondrial dysfunction. The method comprises administering to the mammal an effective amount of an aromatic cationic peptide, the effective amount of the aromatic cationic peptide comprising
At least one net positive charge,
At least 3 amino acids,
Up to about 20 amino acids,
The relationship between the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r), where 3 p _m is the largest number less than r + 1, and p _t can also be 1 if a is 1 Except that 3a has a maximum number less than p _t +1, it has a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p _t ).

Aromatic-cationic peptides include, but are not limited to, the following exemplary peptides.
H-Phe-D-Arg Phe -Lys-Cys-NH 2,
D-Arg-Dmt-Lys-Trp-NH ₂ ,
D-Arg-Trp-Lys-Trp-NH ₂ ,
D-Arg-Dmt-Lys-Phe-Met-NH ₂ ,
HD-Arg-Dmt-Lys (N ^α Me) -Phe-NH ₂ ,
H-D-Arg-Dmt- Lys-Phe (NMe) -NH 2,
HD-Arg-Dmt-Lys (N ^α Me) -Phe (NMe) -NH ₂ ,
^{H-D-Arg (N α} Me) -Dmt (NMe) -Lys (N α Me) -Phe (NMe) -NH 2,
D-Arg-Dmt-Lys-Phe-Lys-Trp-NH ₂ ,
D-Arg-Dmt-Lys- Dmt-Lys-Trp-NH 2,
D-Arg-Dmt-Lys-Phe-Lys-Met-NH ₂ ,
D-Arg-Dmt-Lys-Dmt-Lys-Met-NH ₂ ,
H-D-Arg-Dmt- Lys-Phe-Sar-Gly-Cys-NH 2,
HD-Arg-Arg [CH ₂ -NH] Dmt-Lys-Phe-NH ₂ ,
HD-Arg-Dmt-mt [CH ₂ -NH] Lys-Phe-NH ₂ ,
H-D-Arg-Dmt- LysΨ [CH 2 -NH] Phe-NH 2, and H-D-Arg-Dmt- Ψ [CH 2 -NH] LysΨ [CH 2 -NH] Phe-NH 2,
Tyr-D-Arg-Phe-Lys-NH ₂ ,
2 ', 6'-Dmt-D -Arg-Phe-Lys-NH 2,
Phe-D-Arg-Phe- Lys-NH 2,
Phe-D-Arg-Dmt- Lys-NH 2,
D-Arg-2'6'Dmt-Lys- Phe-NH 2,
H-Phe-D-Arg- Phe-Lys-Cys-NH 2,
Lys-D-Arg-Tyr- NH 2,
D-Tyr-Trp-Lys-NH ₂ ,
Trp-D-Lys-Tyr-Arg-NH ₂ ,
Tyr-His-D-Gly-Met,
Tyr-D-Arg-Phe-Lys-Glu-NH ₂ ,
Met-Tyr-D-Lys-Phe-Arg,
D-His-Glu-Lys-Tyr-D-Phe-Arg,
Lys-D-Gln-Tyr- Arg-D-Phe-Trp-NH 2,
Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His,
Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH ₂ ,
Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH ₂ , Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys,
Lys-Trp-D-Tyr- Arg-Asn-Phe-Tyr-D-His-NH 2, Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys, Asp-D-Trp -Lys-Tyr-D-His- Phe-Arg-D-Gly-Lys-NH 2,
D-His-Lys-Tyr- D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH 2,
Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe,
Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe,
Phe-Phe-D-Tyr- Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH 2,
Phe-Tyr-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr,
Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys,
Glu-Arg-D-Lys- Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH 2,
Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-P-Phe-Tyr-D-Arg-Gly,
D-Glu-Asp-Lys- D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH 2,
Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-P-Ly-Phe,
His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH ₂ , Gly-Ala-Lys -Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp, and Thr-Tyr-Arg- D-Lys-Trp-Tyr- Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH 2,
(Atn) Dap is β- anthraniloyl -L-alpha, a β- diaminopropionic acid, Dmt-D-Arg-Phe- (atn) Dap-NH 2,
(Dns) Dap is β- dansyl -L-alpha, a β- diaminopropionic acid, Dmt-D-Arg-Phe- (dns) Dap-NH 2,
Dmt-D-Arg-Ald-Lys-NH ₂ , wherein Ald is β- (6'-dimethylamino-2'-naphthoyl) alanine,
Dmt-D-Arg-Phe-Lys-Ald-, wherein Ald is .beta .- (6'-dimethylamino-2'-naphthoyl) alanine and D-Arg-Tyr-Lys-Phe-NH ₂
NH _2, as well as one or more of D-Arg-Tyr-Lys- Phe-NH 2.

  In some embodiments, peptides useful in the methods of the present technology are peptides having a tyrosine residue or a tyrosine derivative. In some embodiments, derivatives of tyrosine include 2'-methyltyrosine (Mmt), 2 ', 6'-dimethyltyrosine (2'6'Dmt), 3', 5'-dimethyltyrosine (3'5) 'Dmt), N, 2', 6'-trimethyltyrosine (Tmt), and 2'-hydroxy-6'-methyltyrosine (Hmt).

In one embodiment, the peptide has the formula Tyr-D-Arg-Phe-Lys-NH ₂ (referred to herein as SS-01). SS-01 has three net positive charges contributed by the amino acids tyrosine, arginine and lysine and has two aromatic groups contributed by the amino acids phenylalanine and tyrosine. Tyrosine SS-01 has the formula 2 ', (herein referred to as SS-02) 6'-Dmt- D-Arg-Phe-Lys-NH 2 modified to produce a compound having a , 2 ', 6'- dimethyltyrosine etc. may be derivatives of tyrosine.

In a preferred embodiment, the N-terminal amino acid residue is arginine. Examples of such peptides, D-Arg-2 ', is _{6'Dmt-Lys-Phe-NH 2} ( referred to as SS-31 in this specification).

In another embodiment, the N-terminal amino acid is phenylalanine or a derivative thereof. In some embodiments, derivatives of phenylalanine include 2'-methyl phenylalanine (Mmp), 2 ', 6'-dimethyl phenylalanine (Dmp), N, 2', 6'-trimethyl phenylalanine (Tmp), and 2 '-Hydroxy-6'-methyl phenylalanine (Hmp) is mentioned. An example of such a peptide is Phe-D-Arg-Phe-Lys-NH ₂ (referred to herein as SS-20). In one embodiment, the amino acid sequence of SS-02 is rearranged such that Dmt is not at the N-terminus. Examples of the aromatic cationic peptide has the formula D-Arg-2 ', having a _{6'Dmt-Lys-Phe-NH 2} (SS-31).

In another embodiment, the aromatic-cationic peptide has the formula Phe-D-Arg-Dmt-Lys-NH ₂ (referred to herein as SS-30). Alternatively, the N-terminal phenylalanine can be a derivative of phenylalanine, such as 2 ', 6'-dimethylphenylalanine (2'6' Dmp). At amino acid position 1 2 ', the SS-01 containing 6'-dimethyl-phenylalanine, wherein 2', having a 6'-Dmp-D-Arg- Dmt-Lys-NH 2.

In some embodiments, the aromatic-cationic peptide is 2 ', 6'-Dmt-D-Arg-Phe- (atn), wherein (atn) Dap is β-anthranyloyl-L-α, β-diaminopropionic acid ) Dap-NH ₂ (SS-19), 2 ', 6'-Dmt-D-Arg-Ald-Lys-NH ₂ (SS) wherein Ald is β- (6'-dimethylamino-2'-naphthoyl) alanine -36), 2 ′, 6′-Dmt-D-Arg-Phe-Lys-Ald-NH _{2 wherein} Ald is β- (6′-dimethylamino-2′-naphthoyl) alanine
(SS-37), D-Arg-Tyr-Lys-Phe-NH ₂ (SPI-231), and (dns) Dap is β-dansyl-L-α, β-diaminopropionic acid (SS-17) 2 ', including 6'-Dmt-D-Arg- Phe- (dns) Dap-NH 2.

The peptides described herein and their derivatives can further include functional analogs. A peptide is considered as a functional analogue if the analogue has the same function as the described peptide. Analogs may be, for example, substitution variants of the peptide, wherein one or more amino acids are replaced by another amino acid. Preferred substitution variants of the peptide include conservative amino acid substitutions. Amino acids can be grouped according to their physicochemical characteristics as follows.
(A) Nonpolar amino acid: Ala (A) Ser (S) Thr (T) Pro (P) Gly (G) Cys (C),
(B) Acidic amino acid: Asn (N) Asp (D) Glu (E) Gln (Q), (c) Basic amino acid: His (H) Arg (R) Lys (K),
(D) hydrophobic amino acid: Met (M) Leu (L) Ile (I) Val (V), and (e) aromatic amino acid: Phe (F) Tyr (Y) Trp (W) His (H).

The substitution of an amino acid in a peptide by another amino acid in the same group is termed a conservative substitution and may preserve the physicochemical characteristics of the original peptide. In contrast, substitution of an amino acid in a peptide by another amino acid of a different group is generally more likely to alter the characteristics of the original peptide. Non-limiting examples of analogs useful in the practice of the present technology include, but are not limited to, the aromatic-cationic peptides shown in Table 5.
Table 5. Examples of peptide analogues
Cha = Cyclohexylalanine

Under certain circumstances, it may be advantageous to use a peptide that also has opioid receptor agonist activity. Examples of analogs useful in the practice of the present technology include, but are not limited to, the aromatic-cationic peptides shown in Table 6.
Table 6. Peptide analogs having opioid receptor agonist activity
Dab = diaminobutyric acid Dap = diaminopropionic acid Dmt = dimethyltyrosine Mmt = 2′-methyltyrosine Tmt = N, 2 ′, 6′-trimethyltyrosine Hmt = 2′-hydroxy, 6′-methyltyrosine L-α, β-diaminopropionic acid atnDap = β-anthranyloyl-L-α, β-diaminopropionic acid Bio = biotin

Additional peptides having opioid receptor agonist activity include 2 ', 6'-Dmt-D-Arg-Ald-Lys-NH, where Ald is .beta .- (6'-dimethylamino-2'-naphthoyl) alanine ₂ (SS-36), and 2 ', 6'-Dmt-D-Arg-Phe-Lys-Ald-NH ₂ (SS-, wherein Ald is β- (6'-dimethylamino-2'-naphthoyl) alanine. 37).

  The peptide having mu-opioid receptor agonist activity is typically a peptide having a tyrosine residue or a tyrosine derivative at the N-terminus (i.e., the first amino acid position). Preferred derivatives of tyrosine include 2'-methyltyrosine (Mmt), 2 ', 6'-dimethyltyrosine (2'6'-Dmt), 3', 5'-dimethyltyrosine (3'5'Dmt), N, 2 ', 6'-trimethyltyrosine (Tmt), and 2'-hydroxy-6'-methyltyrosine (Hmt).

  Peptides that do not have mu-opioid receptor agonist activity generally do not have a tyrosine residue or a derivative of tyrosine at the N-terminus (i.e., amino acid position 1). The N-terminal amino acid may be any naturally occurring or non-naturally occurring amino acid other than tyrosine. In one embodiment, the N-terminal amino acid is phenylalanine or a derivative thereof. Exemplary derivatives of phenylalanine include 2'-methyl phenylalanine (Mmp), 2 ', 6'-dimethyl phenylalanine (2', 6'-Dmp), N, 2 ', 6'-trimethyl phenylalanine (Tmp), And 2'-hydroxy-6'-methyl phenylalanine (Hmp).

  The amino acids of the peptides shown in Tables 5 and 6 can be either L- or D-configured.

  In some embodiments, the aromatic-cationic peptide comprises at least one arginine and / or at least one lysine residue. In some embodiments, arginine and / or lysine residues function as electron acceptors and are involved in proton coupled electron transport. Additionally or alternatively, in some embodiments, the aromatic-cationic peptide comprises a sequence that results in a "charge-ring-charge-ring" configuration such as that present in SS-31. Additionally or alternatively, in some embodiments, the aromatic-cationic peptide comprises thiol-containing residues such as cysteine and methionine. In some embodiments, a peptide comprising a thiol containing residue donates electrons directly and reduces cytochrome c. In some embodiments, the aromatic-cationic peptide comprises a cysteine at the N-terminus and / or C-terminus of the peptide.

In some embodiments, peptide multimers are provided. For example, in some embodiments, dimers such as Phe-D-Arg-Phe-Lys-Phe-D-Arg-Phe-Lys, which are SS-20 dimers, are provided. In some embodiments, the dimer is a SS-31 dimer, D-Arg-2 ', 6'-Dmt-Lys-Phe-D-Arg-2', 6'-Dmt-Lys-Phe is -NH _2. In some embodiments, the multimer is a trimer, tetramer, and / or pentamer. In some embodiments, the multimer comprises a combination of different monomeric peptides (eg, an SS-20 peptide linked to an SS-31 peptide). In some embodiments, these longer analogs are useful as therapeutic molecules.

In some embodiments, the aromatic-cationic peptide described herein comprises all levorotatory (L) amino acids.
Peptide synthesis

  The peptides may be synthesized by any of the methods known in the art. Suitable methods for chemically synthesizing proteins are described, for example, by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984) and Methods Enzymol. , 289, Academic Press, Inc, New York (1997).

One way to stabilize the peptide against enzymatic degradation is to replace the L-amino acid with a D-amino acid at the peptide bond undergoing cleavage. Aromatic cationic peptide analogs are prepared to contain one or more D-amino acid residues in addition to the already present D-Arg residues. Another way to prevent enzymatic degradation is to N-methylate the alpha-amino group at one or more amino acid residues of the peptide. This will prevent peptide bond cleavage by any peptidase. As an example, HD-Arg-Dmt-Lys (N ^α Me) -Phe-NH ₂ , HD-Arg-Dmt-Lmt-Phe (NMe) -NH ₂ , HD-Arg-Dmt- ^{Lys (N α Me) -Phe (} NMe) -NH 2, and ^{H-D-Arg (N α} Me) -Dmt (NMe) -Lys (N α Me) -Phe (NMe) -NH 2 and the like. N ^α -methylated analogues can be expected to have lower hydrogen bonding ability and to have improved intestinal permeability.

An alternative way to stabilize the peptide amide bond (-CO-NH-) against enzymatic degradation is to replace it with a reducing amide bond (Ψ [CH ₂ -NH]). This can be accomplished using a reductive alkylation reaction between the Boc-amino acid-aldehyde and the amino group of the N-terminal amino acid residue of the growing peptide chain in solid phase peptide synthesis. Reduced peptide bonds are expected to result in improved cell permeability due to the reduced hydrogen bonding ability. As an example, HD-Arg-Ψ [CH ₂ -NH] Dmt-Lys-Phe-NH ₂ , H-D-Arg-Dmt-Ψ [CH ₂ -NH] Lys-Phe-NH ₂ , H- like D-Arg-Dmt-LysΨ [ CH 2 -NH] Phe-NH 2, H-D-Arg-Dmt-Ψ [CH 2 -NH] LysΨ [CH 2 -NH] Phe-NH 2 .

Cholesterol Cholesterol plays various key physiological roles in cells. Structurally, cholesterol is known to increase membrane order and decrease lipid membrane fluidity (Borchman, Cenedella, & Lamba, 1996, Colell, 2003). Functionally, cholesterol is essential for the production of steroidogenic hormones, bile acids, and vitamin D (Ikonen, 2008, Lucken-Ardjomande, Montessuit, & Martinou, Cell Death Differ. 15 (3): 484-93 ( 2008)).

  Despite the wide variety of different roles of cholesterol in cells, mitochondria are typically described as poor in cholesterol. Healthy mitochondria only maintain about 0.5-3% of the total cellular load of cholesterol to maintain essential functions (Van Meer, Voelker, & Feigenson, 2008). However, many different diseases are onset, eg Alzheimer's disease, cancer, atherosclerosis, fibrosis, insulin resistance, ischemia reperfusion injury, steatohepatitis, Niemann's pick disease, pulmonary fibrosis, and metabolic function The hallmark of conditions such as failure has been shown to have high mitochondrial cholesterol levels and altered mitochondrial cholesterol homeostasis (Colell et al., 2009, Fernandez et al., 2009, Garcia-Ruiz et al. , 2009, Mei et al., 2012, Montero et al., 2008, Musso et al., 2013, Ning et al., 2009, Rouslin et al., 1982, Yu et al., 2005, B. sch et al., 2011). All of these diseases have been shown to have significant mitochondrial dysfunction and significant lipid peroxidation. Mitochondrial dysfunction includes, but is not limited to, decreased respiration, increased reactive oxygen species production, and increased lipid peroxidation.

  High mitochondrial cholesterol adversely affects mitochondrial function, but its mechanism is not yet known. Mitochondria incubated with cholesterol show marked morphological changes (Echegoyen et al., 1993, Yu et al., 2005). Cholesterol exposure results in mitochondrial swelling and lack of hemorrhoid structure (intramitochondrial membrane (IMM) invagination, which is characteristic of mitochondrial structure). Lupus is essential for mitochondrial function in order to increase the functional surface area of IMM and produce more ATP (Mannella, 2006). In addition, incubation with mitochondrial cholesterol shows a marked decrease in mitochondrial respiration and the ability to phosphorylate adenosine diphosophate (ADP) to become adenosine trisophosophate (ATP) (Echegoyen et al., 1993) Rogers et al., 1979, Yu et al., 2005). In addition, an increase in mitochondrial cholesterol results in an increase in the production of reactive oxygen species (ROS) such as superoxide (Mei et al., 2012). ROS is known to cause oxidative damage to lipids, proteins and DNA (Paradies et al., 2000, Paradies et al., 2001, Paradies et al., 2002, Rogers et al., 1979). . As a result, high ROS levels can lead to severe cell damage. Cholesterol exposure also reduces the electrical capacitance (ie, membrane potential) required to perform essential functions throughout the IMM. (Mei et al., 2012).

Lipids Cardiolipin is an important component of the inner mitochondrial membrane and constitutes about 20% of the total lipid component. In mammalian cells, cardiolipin is found almost exclusively in the inner mitochondrial membrane and is essential for the optimal function of enzymes involved in mitochondrial metabolism.

  Cardiolipin is a species of diphosphatidyl glycerol lipid that contains two phosphatidyl glycerols that connect with the glycerol backbone to form a dimeric structure. It has 4 alkyl groups and can carry 2 negative charges. The molecule has a potential for considerable complexity, as there are four individual alkyl chains in cardiolipin. However, in most animal tissues, cardiolipin contains 18 carbon fatty alkyl chains, each of which has 2 unsaturated bonds. The (18: 2) 4-acyl chain configuration is proposed to be an important structural requirement for high affinity of cardiolipin to inner membrane proteins in mammalian mitochondria. However, studies with isolated enzyme preparations show that its importance can vary depending on the protein observed.

  Each of the two phosphates in the molecule can capture one proton. Although it is a target structure, ionization of one phosphate occurs at different levels of acidity than both ionizations at pK1 = 3 and pK2> 7.5. Thus, under normal physiological conditions (pH of about 7.0), the molecule may carry only one negative charge. Hydroxyl groups (-OH and -O-) on phosphoric acid form a bicyclic resonance structure by forming stable intramolecular hydrogen bonds. This structure captures one proton, which leads to oxidative phosphorylation.

  During the oxidative phosphorylation process catalyzed by Complex IV, large amounts of protons are transferred from one side of the membrane to the other, thereby causing a large pH change. Without being bound by theory, cardiolipin has been shown to act as a proton trap within the mitochondrial membrane, strictly localizing the proton pool, and minimizing the pH in the mitochondrial intermembrane space. This function is considered to be due to the unique structure of cardiolipin that protons can be trapped in the bicyclic structure while being negatively charged, as described above. Thus, cardiolipin can act as an electron buffer pool to release or absorb protons to maintain the pH near the mitochondrial membrane.

  In addition, cardiolipin has been shown to play a role in apoptosis. Early events in the apoptotic cascade involve cardiolipin. Cardiolipin-specific oxygenase produces cardiolipin-hydroperoxide, which causes lipids to undergo structural changes. The oxidized cardiolipin then translocates from the inner mitochondrial membrane to the outer mitochondrial membrane where it forms a pore through which it is believed that cytochrome c is released into the cytosol. Cytochrome c can bind to the IP3 receptor to stimulate calcium release, which further promotes the release of cytochrome c. The cells die when cytoplasmic calcium levels reach toxic levels. In addition, extramitochondrial cytochrome c interacts with apoptotic activators to cause formation of apoptosome complexes and activation of the proteolytic caspase cascade.

Another result is that cytochrome c interacts with cardiolipin on the inner mitochondrial membrane with high affinity and is not productive for electron transport, but with a complex with cardiolipin acting as a cardiolipin-specific oxygenase / peroxidase Form. In fact, the interaction of cardiolipin with cytochrome c results in a complex whose normal redox potential is approximately minus (-) 400 mV more negative than that of intact cytochrome c. As a result, the cytochrome c / cardiolipin complex can not receive electrons from mitochondrial complex III, leading to enhanced production of superoxide where disproportionation yields H ₂ O ₂ . The cytochrome c / cardiolipin complex also can not receive electrons from superoxide. In addition, the high affinity interaction of cardiolipin with cytochrome c results in activation of cytochrome c to a cardiolipin-specific peroxidase with selective catalytic activity for the peroxidation of the polyunsaturated molecule cardiolipin . The peroxidase reaction of the cytochrome c / cardiolipin complex is triggered by H ₂ O ₂ as an oxidation equivalent source. Ultimately, this activity results in the accumulation of cardiolipin oxidation products, mainly cardiolipin-OOH, and their reduction products cardiolipin-OH. As mentioned above, oxygenated cardiolipin species have been shown to play a role in mitochondrial membrane permeabilization and release of pro-apoptotic factors (including cytochrome c itself) into the cytosol. For example, Kagan et al., Which is incorporated herein by reference in its entirety. , Advanced Drug Delivery Reviews, 61 (2009) 1375-1385, Kagan et al. , Mol. Nutr. Food Res. 2009 January, 53 (1): 104-114.

Cytochrome c is a globular protein and its main function is to act as an electron carrier from complex III (cytochrome c reductase) to complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain. The prosthetic heme group binds to cytochrome c at Cys14 and Cys17 and further binds to two coordinating axis ligands, His18 and Met80. The sixth coordination that binds to Met80 prevents Fe from interacting with other ligands such as, for example, O ₂ , H ₂ O ₂ , NO.

The pool of cytochrome c is distributed in the intermembrane space and the remainder is associated with IMM through both electrostatic and hydrophobic interactions. Cytochrome c is a highly cationic protein (8+ net charge at neutral pH) that can loosely bind to the anionic phospholipid cardiolipin on the IMM through electrostatic interactions. And, as mentioned above, cytochrome c can also bind tightly to cardiolipin through hydrophobic interactions. This tight binding of cytochrome c to cardiolipin results from the elongation of the acyl chain of cardiolipin which extends from the lipid membrane into the hydrophobic channel within cytochrome c (Tuominen et al., 2001, Kalanxhi & Wallace, 2007, Sinabaldi et al. , 2010). This leads to the rupture of the Fe-Met 80 bond in the cytochrome c heme pocket, resulting in a change in the heme environment as indicated by the loss of the negative cotton peak in the Soure band region (Sinabaldi et al., 2008). This also leads to the exposure of heme Fe to H ₂ O ₂ and NO.

Natural cytochrome c has poor peroxidase activity due to its sixth coordination. However, upon hydrophobic attachment to cardiolipin, cytochrome c undergoes a structural change that disrupts the Fe-Met 80 coordination and increases the exposure of heme Fe to H ₂ O ₂ , and cytochrome c is predominantly cardiolipin. As a substrate, the electron carrier switches to peroxidase (Vladimirov et al., 2006, Basova et al., 2007). As mentioned above, cardiolipin peroxidation results in the altered mitochondrial membrane structure and the release of cytochrome c from IMM to cause caspase-mediated cell death.

Prophylactic and Therapeutic Use of Aromatic-Cationic Peptides Demonstration of the biological effects of aromatic-cationic peptide-based therapeutics. In various embodiments, suitable in vitro or in vivo assays are performed to determine the particular aromatic-cationic peptide-based therapeutic and whether its administration is indicated for treatment. In various embodiments, in vitro assays are performed using representative animal models to determine whether a given aromatic-cationic peptide-based therapeutic exerts a desired effect in the prevention or treatment of a disease. Can. Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to, rats, mice, chickens, pigs, cows, monkeys, rabbits and the like prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art can be used prior to administration to human subjects.

  How to prevent. In one aspect, the present technology provides a method for preventing a cholesterol-induced disease or condition in a subject by administering to the subject an aromatic-cationic peptide that prevents the onset or progression of the disease or condition. In prophylactic applications, pharmaceutical compositions or medicaments of aromatic-cationic peptides are intermediates presented during the onset of the biochemical, histologic and / or behavioral symptoms of the disease, its complications, and the disease. To a subject susceptible to or at risk of a disease or condition, in an amount sufficient to eliminate or reduce the risk of the disease, or to delay the onset of the disease, including pathological manifestations It is administered. The prophylactic aromatic cationic administration may be performed prior to the manifestation of the symptoms characteristic of the disorder to prevent the disease or disorder or to delay its progression. Suitable compounds can be determined based on the screening assays described above.

  Method of treatment. Another aspect of the present technology includes methods of treating a disease in a subject for therapeutic purposes. In therapeutic applications, the composition or medicament is sufficient to cure the symptoms of the disease, reduce its severity, or at least partially arrest its symptoms, including its complications in the onset of the disease and intermediate pathological manifestations. An amount is administered to a subject susceptible to or already suffering from such a disease.

  In one aspect, the present disclosure reduces the number of mitochondria undergoing cholesterol-induced mitochondrial permeability transition (MPT), or a mammal requiring it to prevent cholesterol-induced mitochondrial permeability transition Provided is a method of performing in an animal, the method comprising administering to the mammal an effective amount of one or more of the aromatic-cationic peptides described herein.

  In yet another aspect, the present disclosure provides a method of reducing cholesterol-induced oxidative damage in a subject in need thereof, the method comprising an effective amount of one or more herein Administering the described aromatic cationic peptide to a mammal. In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of reducing cholesterol-induced ROS production in a subject in need thereof.

  In one aspect, the disclosure provides a method of treating, preventing or ameliorating cholesterol-induced mitochondrial dysfunction in a subject in need thereof, the method comprising an effective amount of one or more of the present methods Administration of the aromatic-cationic peptide described in the specification to a mammal.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing cholesterol induced loss of membrane potential in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing cholesterol-induced reduction of cellular respiration in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing cholesterol-induced reduction in the rate of ATP synthesis in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptide of the present technology is a subject in need thereof to treat, ameliorate or reverse cholesterol-induced mitochondrial damage, eg, loss of lupus formation Useful in the methods performed in

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of promoting glutathione (GSH) transport to mitochondria in a subject in need thereof. In certain embodiments, the aromatic-cationic peptide of the present technology treats, ameliorates, or reverses the blockade of cholesterol-induced mitochondrial GSH transport in a subject in need thereof Useful in the method.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing cholesterol-induced cytochrome c peroxidase activity in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing inhibition or reversal of cholesterol-induced damage to cytochrome c in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of performing cholesterol-induced cardiolipin peroxidation in a subject in need thereof.

  In some embodiments, the aromatic-cationic peptides of the present technology are useful in methods of modulating mitochondrial cholesterol levels. In certain embodiments, the aromatic-cationic peptides of the present technology are useful in methods of preventing or treating any cell damage caused by mitochondrial cholesterol overload.

Thus, in some embodiments, the aromatic cationic peptides disclosed herein (D-Arg-2 ', 6'-Dmt-Lys-Phe-NH 2, Phe-D-Arg-Phe-Lys- NH ₂ or an acetate, tartrate, or a pharmaceutically acceptable salt thereof such as trifluoroacetate, etc.) is administered to a subject in need thereof. Without being bound by theory, the peptide contacts the (e.g., target) cytochrome c, cardiolipin, or both, and blocks cholesterol-induced cardiolipin-cytochrome c interaction, resulting in cardiolipin / cytochrome c complex Inhibits the body's cholesterol-induced oxygenase / peroxidase activity, inhibits cholesterol-induced cardiolipin-hydroperoxide formation, inhibits cholesterol-induced translocation of cardiolipin to the outer membrane, and / or induces cholesterol of cytochrome c from IMM It is believed to inhibit sexual release. Additionally or alternatively, in some embodiments, the aromatic-cationic peptides disclosed herein comprise one or more of the following features or functions: (1) cell-permeability, targeting the inner mitochondrial membrane, (2) selectively binding to cardiolipin through electrostatic interactions promoting the interaction of the peptide with cytochrome c, (3) release And (4) protect the hydrophobic heme pocket of cytochrome c, and / or inhibit cardiolipin from disrupting Fe-Met 80 binding, and interacting with cytochrome c that is loosely or tightly bound to cardiolipin (5) promote π-π * interaction with heme porphyrin, (6) inhibit the activity of cytochrome c peroxidase, (7) promote the kinetics of reduction of cytochrome c, (8) cytochrome induced by cardiolipin c Prevents the inhibition of reduction, (9) Promotes electron flux in mitochondrial electron transport chain and ATP synthesis. In some embodiments, the ability of the peptide to enhance electron transport does not correlate with the ability of the peptide to inhibit cholesterol-induced peroxidase activity of the cytochrome c / cardiolipin complex. Thus, in some embodiments, the administered peptide inhibits, retards or reduces the cholesterol-induced interaction of cardiolipin with cytochrome c. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces cholesterol-induced formation of the cytochrome c / cardiolipin complex. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces the cholesterol-induced oxygenase / peroxidase activity of the cytochrome c / cardiolipin complex. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards, or reduces the cholesterol-induced increase in cardiolipin peroxidation. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces cholesterol-induced increase in apoptosis.

Additionally or alternatively, in some embodiments, the method relates to increasing cytochrome c reduction in a sample comprising cytochrome c and cholesterol, wherein the sample comprises an effective amount of an aromatic-cationic peptide, or acetate, tartrate tartrate Contacting with a salt or a salt thereof such as trifluoroacetate salt. Additionally or alternatively, in some embodiments, the method relates to enhancing electron diffusion through cytochrome c in a sample comprising cytochrome c and cholesterol, comprising contacting the sample with an effective amount of an aromatic cationic peptide Including. Additionally or alternatively, in some embodiments, the method relates to enhancing the electronic capacity in cytochrome c in a sample comprising cytochrome c and cholesterol, comprising contacting the sample with an effective amount of an aromatic-cationic peptide . Additionally or alternatively, in some embodiments, the method relates to inducing a novel π-π interaction around cytochrome c in a sample comprising cytochrome c and cholesterol, the sample having an effective amount of aromatic cationic Including contacting with a peptide. In some embodiments, the aromatic cationic peptides, D-Arg-2 ', including 6'-Dmt-Lys-Phe- NH 2. Additionally or alternatively, in some embodiments, the aromatic cationic peptide comprises Phe-D-Arg-Phe- Lys-NH 2. In some embodiments, the method determines whether the sample is an aromatic cationic peptide (eg, D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ or Phe-D-Arg-Phe-Lys- Contact with NH ₂ ) and cardiolipin. In some embodiments, the method comprises contacting the sample with cardiolipin.

  The aromatic-cationic peptide described herein is useful for the prevention or treatment of a disease. Specifically, the disclosure relates to administering an aromatic-cationic peptide as described herein to a subject at risk of (or susceptible to) a disease characterized by cholesterol-induced mitochondrial dysfunction. Provide both prophylactic and therapeutic methods of treatment. Thus, the method provides for the prevention and / or treatment of a disease characterized by cholesterol-induced mitochondrial dysfunction in a subject by administering an effective amount of an aromatic-cationic peptide to the subject in need thereof. .

  Oxidative damage. The peptides disclosed herein are useful for reducing oxidative damage in mammals in need thereof. A mammal in need of reducing oxidative damage is a mammal undergoing a disease, condition, or treatment with oxidative damage. Typically, oxidative damage is caused by free radicals such as reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). Examples of ROS and RNS include hydroxyl radical, superoxide anion radical, nitric oxide, hydrogen, hypochlorous acid (HOCl), and peroxynitrite anion. Oxidative damage is considered "reduced" if the amount of oxidative damage, excised organs or cells in a subject is reduced after administration of an effective amount of the above-mentioned aromatic-cationic peptide. Typically, the oxidative damage is at least about 10%, at least about 25%, at least about 50%, at least about 75%, as compared to a control subject that has not been treated with peptide. Or reduced by at least about 90%.

  In some embodiments, the subject to be treated can be a mammal having a disease or condition associated with oxidative damage. Oxidative damage can occur in any cell, tissue or organ of a mammal. In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Alzheimer's disease and the like.

  In some embodiments, the mammal can be treated with oxidative damage. For example, a mammal may receive reperfusion. Reperfusion refers to the restoration of blood flow to any organ or tissue in which blood flow has been reduced or blocked. Reconstruction of blood flow during reperfusion leads to respiratory burst and formation of free radicals.

  In one embodiment, the mammal may have reduced or blocked blood flow due to hypoxia or ischemia. Loss or severe reduction of blood supply during hypoxemia or ischemia may be due to, for example, thromboembolic stroke, coronary atherosclerosis, or peripheral vascular injury. Many organs and tissues are exposed to ischemia or hypoxia. Examples of such organs include the brain, heart, kidney, intestine and prostate. The affected tissue is typically a muscle such as cardiac muscle, skeletal muscle or smooth muscle. For example, myocardial ischemia or hypoxia is generally caused by atherosclerotic or thrombotic occlusion leading to reduced or lost oxygen delivery to cardiac tissue by the cardiac arterial and capillary blood supplies. Such cardiac ischemia or hypoxia can cause pain and necrosis of the affected myocardium and can ultimately lead to heart failure.

  The method can also be used in reducing oxidative damage associated with any neurodegenerative disease or condition. Neurodegenerative diseases can affect any cell, tissue or organ of the central and peripheral nervous system. Examples of such cells, tissues and organs include brain, spinal cord, neurons, ganglia, Schwann cells, astrocytes, oligodendrocytes and microglia. The neurodegenerative condition can be an acute condition such as stroke or traumatic brain injury or spinal cord injury. In another embodiment, the neurodegenerative disease or condition may be a chronic neurodegenerative condition. In chronic neurodegenerative conditions, free radicals can cause, for example, damage to proteins. An example of such a protein is amyloid beta-protein. Examples of chronic neurodegenerative diseases accompanied by free radical damage include Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (also known as Lugueric disease).

  Mitochondrial permeability transition. The peptides disclosed herein are also useful in treating any disease or condition involving mitochondrial permeability transition (MPT). Such diseases and conditions include, but are not limited to, tissue or organ ischemia and / or reperfusion, hypoxia, and any of a number of neurodegenerative diseases. Mammals in need of inhibition or prevention of MPT are mammals suffering from these diseases or conditions.

Apoptosis. The peptides disclosed herein are useful in treating diseases or conditions that involve apoptosis. Exemplary diseases or conditions include colon cancer, glioma, liver cancer, neuroblastoma, leukemia and lymphoma, and cancers such as prostate; myasthenia gravis, systemic lupus erythematosus, inflammatory disease, bronchial asthma, inflammation Enteric diseases, autoimmune diseases such as lung inflammation; virus infections such as adenovirus and baculovirus and HIV-AIDS; Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, retinitis pigmentosa, and nerves such as epilepsy Degenerative diseases; Hematological diseases such as aplastic anemia, myelodysplastic syndrome, T CD4 + lymphopenia, and G6 PD deficiency Tissue damage, but is not limited thereto. Thus, in some embodiments, the aromatic cationic peptides disclosed herein (D-Arg-2 ', 6'-Dmt-Lys-Phe-NH 2, Phe-D-Arg-Phe-Lys- NH ₂ or an acetate, tartrate, or a pharmaceutically acceptable salt thereof such as trifluoroacetate is administered to a subject (eg, a mammal such as human) in need thereof. As described above, the peptide contacts (e.g., target) cytochrome c, cardiolipin, or both to block cholesterol-induced cardiolipin-cytochrome c interaction and inhibit cholesterol-induced cardiolipin-hydroperoxide formation It is believed that it inhibits cholesterol-induced translocation of cardiolipin to the outer membrane and / or inhibits cholesterol-induced oxygenase / peroxidase activity. Thus, in some embodiments, the administered peptide inhibits, retards or reduces the cholesterol-induced interaction of cardiolipin with cytochrome c. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces cholesterol-induced formation of the cytochrome c / cardiolipin complex. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces the cholesterol-induced oxygenase / peroxidase activity of the cytochrome c / cardiolipin complex. Additionally or alternatively, in some embodiments, the administered peptide inhibits, retards or reduces cholesterol-induced apoptosis.

  Cardiolipin oxidation. The peptides disclosed herein are useful for preventing, inhibiting or reducing cardiolipin oxidation. Cardiolipin is a unique phospholipid found almost exclusively at the inner mitochondrial membrane and at the contact site connecting the outer and inner membranes. The cardiolipin domains are essential for proper chewing formation and they are involved in organizing respiratory complexes into higher order hypercomplexes to facilitate electron transfer. Cardiolipin is also essential to keep cytochrome c close to the respiratory complex for efficient electron transfer. The positively charged cytochrome c interacts with highly anionic cardiolipin via electrostatic interactions.

  This cardiolipin-cytochrome c interaction is attenuated by cardiolipin peroxidation, and loss of free cytochrome c to the cytosol causes caspase-dependent apoptosis (Shidoji et al., 1999, Biochem Biophys Res Commun 264, 343-347. ). Lipid membrane remodeling occurs during apoptosis and phosphatidylserine moves from the inner to the outer apex of the plasma membrane. In a similar manner, cardiolipin and its metabolites can be transferred from mitochondria to other intracellular organelles and to the cell surface (Sorice et al., 2004, Cell Death Differ 11, 1131-3145).

Aromatic cationic peptide (2 ', 6'-Dmt-D-Arg-Phe-Lys-NH ₂ (SS-02), Phe-D-Arg-Phe-Lys-NH ₂ (SS −20), and D-Arg-2 ′, 6′-Dmt-Lys-Phe-NH ₂ (SS-31), or a pharmaceutically acceptable salt thereof such as acetate, tartrate, and trifluoroacetate Salts and the like, but not limited thereto, are useful for preventing cardiolipin peroxidation. Cardiolipin peroxidation is mainly mediated by the peroxidase activity of cytochrome c. About 15-20% of mitochondrial cytochrome c is tightly bound in complex with cardiolipin via hydrophobic interactions, whereby one or more acyl chains of cardiolipin are inserted into the hydrophobic channel of cytochrome c Ru. Insertion of the acyl chain into cytochrome c breaks the coordinate bond between Met80 and heme Fe, exposing the sixth coordination of heme Fe to H ₂ O ₂ , thus oxidizing this enzyme, cardiolipin Is selectively converted to peroxidase that can be catalyzed.

Aromatic cationic peptide (2 ', 6'-Dmt-D-Arg-Phe-Lys-NH ₂ (SS-02), Phe-D-Arg-Phe-Lys-NH ₂ (SS −20), and D-Arg-2 ′, 6′-Dmt-Lys-Phe-NH ₂ (SS-31), or a pharmaceutically acceptable salt thereof such as acetate, tartrate, and trifluoroacetate Salts and the like, but not limited thereto, are cell permeable and selectively target and concentrate in the inner mitochondrial membrane. Aromatic cationic peptides selectively target anionic phospholipids such as cardiolipin, phosphatidylserine, phosphatidic acid, and phosphatidylglycerol, with cardiolipin having the highest affinity with some peptides. In some embodiments, the aromatic-cationic peptide interacts with cytochrome c, which interaction is enhanced in the presence of cardiolipin. In some embodiments, the peptide can penetrate into the cytochrome c protein in the immediate vicinity of heme, and this penetration is enhanced in the presence of cardiolipin.

  Without being bound by theory, by penetrating into the heme environment of cytochrome c, the aromatic-cationic peptide interferes with the structural interaction between cardiolipin and cytochrome c, and Met80-Fe coordination It is highly likely to prevent the bursting of cytochrome c and prevent cytochrome c from becoming peroxidase. These aromatic cationic peptides also increase π-π * interactions in the heme region and promote cytochrome c reduction.

The aromatic-cationic peptides disclosed herein can also enhance mitochondrial respiration, increase oxidative phosphorylation capacity, and reduce mitochondrial reactive oxygen species. As a result, they can protect mitochondrial function, reduce cardiolipin peroxidation and / or inhibit apoptosis.
Niemann-Pick disease

  The peptides disclosed in the present specification include Alzheimer's disease, cancer, atherosclerosis, fibrosis, insulin resistance, ischemia reperfusion injury, Niemann's pick, non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, and metabolism. It is useful for treating diseases or conditions characterized by cholesterol-induced mitochondrial dysfunction, such as dysfunction.

  Niemann-Pick disease refers to a group of hereditary serious metabolic disorders (including Niemann-Pick Type C (NPC)) that allow the accumulation of sphingomyelin in the lysosome. NPC patients can not properly metabolize cholesterol and other lipids in cells. As a result, excess cholesterol accumulates in the mitochondrial membrane of the liver, spleen and nerve cells. In addition, mitochondrial membrane potential and ATP levels are markedly reduced in NPC mouse brain and neurons. It has been shown that reducing the level of cholesterol in mitochondrial membranes using methyl-beta-cyclodextrin may restore the activity of ATP synthase. Yu et al. , J. Biol. Chem. 280: 11731-11739 (2005). Furthermore, NPC neurons exhibit neurite outgrowth disorders, which can be reduced by exogenous ATP. These results indicate that mitochondrial dysfunction and subsequent ATP deprivation may be the cause of neuronal damage in NPC disease. Symptoms of Niemann-Pick disease include difficulty moving the limbs (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly), dementia, unclear and irregular speech (arthria), dysphoria ( Dysphagia), sudden loss of muscle tone that can lead to falls (acute seizures), tremor, difficulty moving the eyes up and down (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia Disease, jaundice, pain, enlargement of the medullary cavity, thinning of cortical bone, distortion of the hip bone, seizures, cherry red spots in the eye, and symptoms of sleep related disorder harm.

Cardiolipin peroxidation and increased oxidative stress may be important processes in the pathology of Niemann-Pick disease. Thus, the aromatic cationic peptides of the present disclosure (2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys-NH 2 (SS- 20), and D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31), or acetate, pharmaceutically acceptable salts such as tartrate and trifluoroacetate, And the like) can be used to treat Niemann-Pick disease by preventing cardiolipin oxidation, oxidative stress, and cell apoptosis in a subject.

Aromatic Cationic Peptides in Electron Transfer Mitochondrial ATP synthesis is triggered by electron flow through the electron transport chain (ETC) of the IMM. Electron flow through the chain can be described as a series of oxidation / reduction processes. Electrons pass from the electron donor (NADH or QH2), through a series of electron acceptors (complexes I to IV), and finally to molecular oxygen which is the final electron acceptor. Cytochrome c, which is loosely associated with IMM, transfers electrons between complexes III and IV.

  Rapid replacement of electrons through ETC is important to prevent short circuits leading to electron efflux and the formation of free radical intermediates. Electron transfer (ET) between the electron donor and the electron acceptor decreases exponentially with the distance between them, and the superexchange ET is limited to 20 Å. Long distance ET can be achieved in a multistep electron hopping process, where the overall distance between donor and acceptor is divided into a series of shorter and thus faster ET steps. In ETC, efficient ET over long distances is supported by strategically localized cofactors along the IMM, including FMN, FeS clusters, and heme. Aromatic amino acids such as Phe, Tyr, and Trp can also facilitate electron transfer to heme through overlapping pi clouds. Amino acids (Tyr, Trp, Cys, Met) having suitable oxidizing ability can act as foundations by acting as intermediate electron carriers. In addition, the hydroxyl group of Tyr can lose protons when transporting electrons, and the presence of a basic group in the vicinity such as Lys can result in a proton-conjugated ET that is even more efficient.

Overexpression of catalase that targets mitochondria (mCAT) has been shown to improve aging (eg, reduce symptoms) and prolong lifespan in mice. Such examples identify chemical compounds that can be “drug target” that can reduce mitochondrial oxidative stress and protect mitochondrial function. Mitochondria are the main source of intracellular reactive oxygen species (ROS), so antioxidants can be confined to mitochondrial DNA, electron transport chain (ETC) proteins, and mitochondrial lipid membranes. Need to be delivered to the mitochondria. In some embodiments, the aromatic-cationic peptide selectively targets and concentrates in the IMM. Some of these peptides contain redox active amino acids that can undergo single electron oxidation and behave as mitochondrial targeted antioxidants. D-Arg-2'6'-Dmt- Tyr-Lys-Phe-NH 2 Peptides disclosed herein, such as, decreased mitochondrial ROS, protect mitochondrial function in cell and animal studies. Recent studies show that this peptide can confer protection from mitochondrial oxidative stress comparable to that observed with mitochondrial catalase overexpression. Radical scavenging is the most commonly used procedure to reduce oxidative stress, but it can be used to enhance electron transfer to reduce electron leakage and improve mitochondrial reduction capabilities. There is a possible mechanism of

  Abundant indirect evidence indicates that oxidative stress contributes to normal aging and the consequences of many of the several major diseases including cardiovascular disease, diabetes, neurodegenerative diseases, and cancer. Oxidative stress is generally defined as an imbalance between prooxidants and antioxidants. However, despite massive scientific evidence to support increased oxidative tissue damage, extensive clinical studies with antioxidants have not shown significant health benefits in these diseases. One of the reasons may be that the available antioxidants can not reach the site of prooxidant production.

  The mitochondrial electron transport chain (ETC) is the major intracellular producer of ROS, and the mitochondria itself is most vulnerable to oxidative stress. Protecting mitochondrial function is thus a prerequisite for preventing cell death caused by mitochondrial oxidative stress. The benefits of overexpressing catalase (mCAT) targeting mitochondria, but not peroxisomes (pCAT), demonstrate the proof that mitochondrial targeting antioxidants are required to overcome the adverse effects of aging. Provided. However, sufficient delivery of chemical antioxidants to the IMM remains difficult.

One peptide analogues, D-Arg-2 ', 6'-Dmt-Tyr-Lys-Phe-NH 2 is to possess endogenous antioxidant potential, which is because the modified tyrosine residue, oxide It is because it is reductive and can be subjected to one-electron oxidation. This peptide can neutralize H ₂ O ₂ , hydroxyl radicals, and peroxynitrite to inhibit lipid peroxidation. The peptides have shown great efficacy in animal models of ischemia reperfusion injury, neurodegenerative diseases and metabolic syndrome.

  The design of the mitochondrial targeting peptide may be one or more of the following actions: (i) elimination of excess ROS, (ii) reduction of ROS production by promoting electron transfer, or (iii) increase in mitochondrial reducibility Incorporate and strengthen. The advantages of peptide molecules are that they can function as redox centers, facilitate electron transfer, or enhance sulfhydryl groups while retaining aromatic cationic motifs that are essential for mitochondrial targeting. It is possible to incorporate natural amino acids.

Mode of Administration and Effective Dose Any method known to those skilled in the art for contacting cells, organs or tissues with aromatic cationic peptides may be used. Suitable methods include in vitro, ex vivo or in vivo methods. In vitro methods typically include cultured samples. For example, cells can be placed in a container (eg, a tissue culture plate) and incubated with the aromatic-cationic peptide under appropriate conditions suitable to obtain the desired result. Suitable incubation conditions can be readily determined by one skilled in the art.

  Ex vivo methods typically involve cells, organs, or tissues excised from a mammal, such as a human. Cells, organs, or tissues can be incubated, for example, with an aromatic-cationic peptide under appropriate conditions. The contacted cells, organs, or tissues are typically returned to the donor, placed in the recipient, or stored for future use. Thus, the aromatic-cationic peptide is generally present in a pharmaceutically acceptable carrier.

  In vivo methods typically involve administering an aromatic cationic peptide such as those described above to a mammal, preferably a human. When used in vivo for therapeutic purposes, the aromatic-cationic peptide is administered to the subject in an effective amount (ie, an amount that has the desired therapeutic effect). The dose and dosing regimen will depend on the degree of injury in the subject, the characteristics of the particular aromatic-cationic peptide used, such as the therapeutic index, the subject, and the subject's medical history. The effective amount can be determined during preclinical or clinical trials by methods well known to the physician and clinician.

  An effective amount of the aromatic-cationic peptide useful in the present methods can be administered to a mammal in need thereof by any of a number of known methods for administering pharmaceutical compounds. The aromatic-cationic peptide can be administered systemically or locally.

  The aromatic cationic peptide may be formulated as a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a salt prepared from a base or acid which is acceptable for administration to a patient, such as a mammal (eg, a mammal that is acceptable for a given dosing regimen) Safety salt). However, it is to be understood that these salts do not necessarily have to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases, and from pharmaceutically acceptable inorganic or organic acids. In addition, when the peptide contains both a basic moiety such as an amine, pyridine or imidazole and an acidic moiety such as a carboxylic acid or tetrazole, zwitterions can be formed, as used herein Included in "salt". As salts derived from pharmaceutically acceptable inorganic bases, ammonium salts, calcium salts, copper salts, ferric salts, ferrous salts, lithium salts, magnesium salts, manganous salts, manganous salts, potassium And salts, sodium salts, zinc salts and the like. As salts derived from pharmaceutically acceptable organic bases, primary, secondary and tertiary amines including substituted amines, cyclic amines, natural amines and the like, such as arginine, betaine, caffeine , Choline, N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine And salts of lysine, methylglucamine, morpholine, piperazine, piperazine (piperadine), polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.As salts derived from pharmaceutically acceptable inorganic acids, boric acid, carbonic acid, hydrohalic acid (hydrobromic acid, hydrochloric acid, hydrofluoric acid or hydroiodic acid), nitric acid, phosphoric acid, sulfamic acid And salts of sulfuric acid. As salts derived from pharmaceutically acceptable organic acids, aliphatic hydroxyl acids (eg, citric acid, gluconic acid, glycolic acid, lactic acid, lactobionic acid, malic acid, and tartaric acid), aliphatic monocarboxylic acids (eg, Acetic acid, butyric acid, formic acid, propionic acid and trifluoroacetic acid), amino acids (for example, aspartic acid and glutamic acid), aromatic carboxylic acids (for example, benzoic acid, p-chlorobenzoic acid, diphenylacetic acid, gentisic acid, hippuric acid, And triphenylacetic acid), aromatic hydroxyl acids (eg, o-hydroxybenzoic acid, p-hydroxybenzoic acid, 1-hydroxynaphthalene-2-carboxylic acid, and 3-hydroxynaphthalene-2-carboxylic acid), ascorbic acid, Dicarboxylic acids (eg fumaric acid, maleic acid, oxalic acid and succinic acid), gum Glucononic acid, mandelic acid, mucic acid, nicotinic acid, pumonic acid, pantothenic acid, sulfonic acid (eg, benzenesulfonic acid, camphosphosulfonic acid, edicilic acid, ethanesulfonic acid, isethionic acid And salts thereof such as methanesulfonic acid, naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2,6-disulfonic acid, and p-toluenesulfonic acid), xinafoic acid and the like. In some embodiments, the salt is acetate. In some embodiments, the salt is a tartrate salt. In addition or alternatively, the salt is trifluoroacetate salt.

  The aromatic-cationic peptides described herein can be incorporated into pharmaceutical compositions for administration alone or in combination to subjects for the treatment or prevention of the disorders described herein. Such compositions typically comprise an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents compatible with drug administration. And absorption delay agents and the like. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoresis, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may be water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or sterile diluents such as other synthetic solvents, Antimicrobial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylene diamine tetraacetic acid, buffers such as acetate, citrate or phosphate, and sodium chloride or glucose And other ingredients such as agents for adjusting tonicity. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For the convenience of the patient or treating physician, the dosage form should have all the necessary equipment (eg, vial of drug, vial of diluent, syringe, and so on) for the treatment course (eg, 7 days of treatment). Can be provided in a kit comprising an injection needle).

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). Be In all cases, the composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage, and should be protected against the contaminating action of microorganisms, such as bacteria and fungi.

  In one embodiment, the aromatic-cationic peptide of the present technology is administered intravenously. For example, the aromatic-cationic peptide may be administered by rapid intravenous bolus injection. In some embodiments, the aromatic-cationic peptide is administered as a constant speed intravenous infusion.

  The aromatic-cationic peptide may also be administered orally, topically, nasally, intramuscularly, subcutaneously or transdermally. In one embodiment, transdermal administration is administered by iontophoresis, in which the charged composition is delivered through the skin by an electrical current.

  Other routes of administration include intracerebroventricular or intrathecal. Intracerebroventricular refers to administration to the ventricular system of the brain. Intrathecal refers to administration to the subarachnoid space of the spinal cord. Thus, in some embodiments, intracerebroventricular or intrathecal administration is used for diseases and conditions that affect organs or tissues of the central nervous system.

  Aromatic cationic peptides may also be administered to mammals by sustained release, as known in the art. Sustained release administration is a drug delivery method to achieve a certain level of drug over a certain period of time. This level is typically measured by serum or plasma concentrations. A description of methods for delivering compounds by controlled release can be found in International PCT Application WO 02/083106, which is incorporated herein by reference in its entirety.

  Any dosage form known in the pharmaceutical art is suitable for administration of the aromatic-cationic peptide. For oral administration, solutions or solids may be used. Examples of dosage forms include tablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the like. The aromatic-cationic peptide can be mixed with a suitable pharmaceutical carrier (vehicle) or excipient as understood by those skilled in the art. Examples of carriers and excipients include starch, milk, certain types of clay, gelatin, lactic acid, stearic acid containing magnesium stearate or calcium stearate or salts thereof, talc, vegetable fat or oil, gum, and Glycol is mentioned.

  For systemic, intracerebroventricular, intrathecal, topical, nasal, subcutaneous, or transdermal administration, dosage forms of the aromatic-cationic peptide may be conventional diluted carriers such as those known in the art, Alternatively, an aromatic cationic peptide may be delivered using an excipient or the like. For example, the dosage form may comprise one or more of stabilizers, surfactants, preferably nonionic surfactants, and optionally salts and / or buffers. The aromatic-cationic peptide may be delivered in the form of an aqueous solution or in a lyophilised form.

  Stabilizers may include, for example, amino acids such as glycine; oligosaccharides such as sucrose, tetralose, lactose, or dextran. Alternatively, the stabilizer may comprise a sugar alcohol such as mannitol. In some embodiments, the stabilizer or combination of stabilizers comprises about 0.1% to about 10% by weight of the weight of the formulated composition.

  In some embodiments, the surfactant is a non-ionic surfactant such as polysorbate. Examples of suitable surfactants include Tween 20, Tween 80; polyethylene glycol or polyoxyethylene poly such as Pluronic F-68 at about 0.001% (w / v) to about 10% (w / v) Oxypropylene glycol is mentioned.

  The salt or buffer may be any salt or buffer, such as, for example, sodium chloride or sodium / potassium phosphate, respectively. In some embodiments, the buffering agent maintains the pharmaceutical composition pH in the range of about 5.5 to about 7.5. Salts and / or buffers are also useful to maintain the osmotic pressure at a level suitable for human or animal administration. In some embodiments, the salt or buffer is present at an approximately isotonic concentration of about 150 mM to about 300 mM

  The dosage form of the aromatic-cationic peptide may further contain one or more conventional additives. Examples of such additives are solubilizers, such as, for example, glycerol; for example, benzalkonium chloride (mixture of quaternary ammonium compounds known as "quaternary"), benzyl alcohol, chlorethon, or chlorobutanol etc. Antioxidants; anesthetics such as morphine derivatives; and isotonic agents as described herein. As a further precaution against oxidation or other spoilage, the pharmaceutical composition may be stored under nitrogen gas in a vial sealed with an impermeable stopper.

  Mammals treated according to the present technology are any mammals including, for example, livestock such as sheep, pigs, cattle and horses; companion animals such as dogs and cats; and laboratory animals such as rats, mice and rabbits It may be In one embodiment, the mammal is a human.

  In some embodiments, the aromatic-cationic peptide is administered to the mammal in an amount effective to reduce the number of mitochondria undergoing MPT or to prevent MPT. The effective amount is determined during preclinical or clinical trials by methods well known to physicians and clinicians.

  The aromatic-cationic peptide can be administered systemically or locally. In one embodiment, the aromatic-cationic peptide is administered intravenously. For example, the aromatic-cationic peptide may be administered by rapid intravenous bolus injection. In one embodiment, the aromatic-cationic peptide is administered as a constant speed intravenous infusion.

  The aromatic-cationic peptide can be injected directly into the coronary artery, for example during angioplasty or coronary artery bypass surgery, or applied on a coronary stent.

  The aromatic-cationic peptide composition can be a carrier, which contains, for example, water, ethanol, a polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof It may be a solvent or dispersion medium. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, ascorbic acid, thiomerasol and the like. Oxidation can be prevented by including glutathione and other antioxidants. In some embodiments, an isotonic agent, for example, a sugar, a polyhydric alcohol such as mannitol, sorbitol, or sodium chloride is included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

  Sterile injectable solutions are prepared by incorporating the active ingredient (s) into a suitable solvent in the required amount, optionally with one or a combination of ingredients listed above, and filter sterilizing be able to. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical preparation method is to add to the active ingredient plus a powder of any further desired ingredient from that solution which has been sterile filtered beforehand. Vacuum drying and lyophilization can be obtained.

  Oral compositions generally include an inert diluent and an edible carrier. For the purpose of oral therapeutic administration, the active compound can be used in combination with excipients, in the form of tablets, troches, or capsules, eg, gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc., binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; magnesium stearate Or lubricants such as Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or compounds such as flavorings such as peppermint, methyl salicylate or orange flavor or compounds of similar properties Both can be contained.

  For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser, which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. Such methods include those described in US Pat. No. 6,468,798.

  Systemic administration of a therapeutic compound as described herein may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the dosage form. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished through the use of a nasal sprayer. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

  Therapeutic proteins or peptides can be formulated in a carrier system. The carrier may be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic peptide is encapsulated in liposomes while maintaining peptide integrity. As one skilled in the art will appreciate, there are various methods for preparing liposomes. (See Lichtenberg et al., Methods Biochem. Anal., 33: 337-462 (1988), Anselem et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (see Reddy, Ann. Pharmacother., 34 (7-8): 915-923 (2000)). The active agent is loaded into particles prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable, or gastric retentive polymers or liposomes. It can also be done. Such particles include nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems. But not limited thereto.

  The carrier can also be a polymer, such as a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic peptide can be embedded in a polymer matrix while maintaining protein integrity. The polymer may be a polypeptide, a protein, or a natural type such as a polysaccharide, or a synthetic type such as poly α-hydroxy acid. Examples include, for example, carriers made of collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA) or a lactic acid glycolic acid copolymer (PGLA). The polymer matrix can be prepared and isolated in various forms and sizes, including microspheres and nanospheres. Polymer dosage forms can lead to long-term therapeutic effects. (See Reddy, Ann. Pharmacother., 34 (7-8): 915-923 (2000)). Polymer dosage forms for human growth hormone (hGH) have been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2: 548-552 (1998)).

  Examples of polymeric microsphere sustained release formulations are described in PCT WO 99/15154 (Tracy et al.), US Pat. Nos. 5,674,534 and 5,716,644 (both Zale et al.), PCT International Publications 96/40073 (Zale et al.) And PCT WO 00/38651 (Shah et al.). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT WO 96/40073 describe polymer matrices containing particles of erythropoietin which are stabilized against aggregation by salts. doing.

  In some embodiments, the therapeutic compound is formulated with a carrier that protects the therapeutic compound, such as a controlled release formulation, including implanted and microencapsulated delivery systems, against rapid clearance from the body. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acids. Such dosage forms can be prepared using known techniques. Substances can also be obtained commercially, for example from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies against cell specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as, for example, those described in US Pat. No. 4,522,811.

  Therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposome delivery systems are known in the art, eg, Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6: 698-708 (1995), Weiner, “Liposomes for Protein Delivery. : Selecting Manufacture and Development Processes, "Immunomethods, 4 (3): 201-9 (1994)", and Gregoriadis, "Engineering Liposomes for Drug Delivery: Progress and Pro lems, "Trends Biotechnol. , 13 (12): 527-37 (1995). Mizguchi et al. , Cancer Lett. 100: 63-69 (1996) describe the use of fusogenic liposomes for delivering proteins to cells both in vivo and in vitro.

  The dose, toxicity, and therapeutic efficacy of the therapeutic agent can be determined, for example, to determine LD50 (dose that is lethal to 50% of the population) and ED50 (dose that is therapeutically effective in 50% of the population) Can be determined by standard pharmaceutical procedures in cell culture or in experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but to the site of diseased tissue that targets such compounds to minimize potential damage to non-diseased cells and thereby reduce side effects. Care should be taken in the design of the delivery system.

  The data obtained from cell culture assays and animal studies can be used in formulating a range of doses for use in humans. The dose of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the formulation employed and the route of administration utilized. For any compound used in the method, the therapeutically effective dose can be estimated from cell culture assays. Doses can be formulated in animal models to achieve a blood plasma concentration range that includes the IC50 as determined in cell culture (ie, the concentration of test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

  Typically, an effective amount of aromatic-cationic peptide sufficient to achieve a therapeutic or prophylactic effect is in the range of about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. is there. Preferably, the dose range is about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example, the dose may be 1 mg / kg body weight or 10 mg / kg body weight daily, every other day, or every three days, or 1-10 mg / kg weekly, biweekly, or every three weeks. In one embodiment, a single dose of peptide is in the range of 0.1 to 10,000 micrograms per kilogram of body weight. In one embodiment, the aromatic-cationic peptide concentration in the carrier is in the range of 0.2-2000 micrograms per milliliter delivered. Exemplary treatments involve administration once daily or once weekly. In therapeutic applications, relatively high doses at relatively short intervals may be sought until the progression of the disease is reduced or stopped, and preferably, the subject shows partial or complete improvement in the symptoms of the disease. There is a case. The patient can then be given a preventive regimen.

In some embodiments, a therapeutically effective amount of an aromatic-cationic peptide may be defined as the concentration of the peptide at 10 ^-12 to 10 ^-6 moles, eg, about 10 ^-7 moles, of the target tissue. This concentration can be delivered with systemic dosing of 0.01-100 mg / kg or equivalent surface area of body surface. The dosing schedule is optimized to maintain therapeutic concentrations in the target tissue, which is most preferably by a single daily or weekly administration, but also continuous administration (eg parenteral injection or transdermal application) Including.

  In some embodiments, the dose of aromatic cationic peptide is provided at about 0.001 to about 0.5 mg / kg / h, preferably about 0.01 to about 0.1 mg / kg / h. In one embodiment, the dose of aromatic cationic peptide is provided at about 0.1 to about 1.0 mg / kg / h, preferably about 0.1 to about 0.5 mg / kg / h. In one embodiment, the dose is provided at about 0.5 to about 10 mg / kg / h, preferably about 0.5 to about 2 mg / kg / h.

  One skilled in the art will appreciate that certain factors, including but not limited to the severity of the disease or disorder, past treatment, the subject's general health and / or age, and other diseases present. It will be understood that it may affect the dose and timing needed to effectively treat. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic composition described herein may comprise a single treatment or a series of treatments.

  Mammals treated according to this method are any mammals including, for example, livestock such as sheep, pigs, cattle and horses; companion animals such as dogs and cats; laboratory animals such as rats, mice and rabbits It may be. In one embodiment, the mammal is a human.

Combination Therapy In some embodiments, the aromatic-cationic peptide may be used in combination with one or more additional therapeutic agents for the prevention, amelioration, or treatment of a medical disease or condition. By way of example and not limitation, treatment of mitochondrial diseases or disorders typically involves taking vitamins and cofactors. In addition, as non-limiting examples, antibiotics, hormones, antineoplastic agents, steroids, immunomodulators, dermatological agents, antithrombotic agents, antianemic agents, and cardiovascular agents may also be administered.

  In one embodiment, the aromatic-cationic peptide is used in combination with one or more cofactors, vitamins, iron chelators, antioxidants, frataxin level modifiers, ACE inhibitors, and β-blockers. By way of example and not limitation, such compounds include CoQ10, levocarnitine, riboflavin, acetyl-L-carnitine, thiamine, nicotinamide, vitamin E, vitamin C, lipoic acid, selenium, beta carotene, biotin, folic acid, Calcium, magnesium, phosphorus, succinate, selenium, creatine, uridine, citratesm prednisone, vitamin K, deferoxamine, deferiprone, idebenone, erythropoietin, 17β estradiol, methylene blue, and BML-210 and compound 106, etc. It may also include one or more of histone deacetylases.

  In one embodiment, the additional therapeutic agent is administered to the subject in combination with the aromatic-cationic peptide to produce a synergistic therapeutic effect. A "synergistic therapeutic effect" refers to a more than additive therapeutic effect produced by the combination of at least two therapeutic agents and beyond that which results separately from the administration of at least two therapeutic agents alone. Thus, the use of low doses of one or more of at least two therapeutic agents for the treatment of a disease or disorder may result in increased therapeutic efficacy and reduced side effects.

  Multiple therapeutic agents (including aromatic-cationic peptides) may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be applied in a single integrated form or in multiple forms (for illustration purposes only, either as a single pill or as two separate pills) ). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between multiple doses may differ from more than 0 weeks to less than 4 weeks. In addition, combination methods, compositions, and dosage forms are not limited to the use of two agents.

  The technology is further illustrated by the following examples, which should not be construed as limiting in any way.

General Methods Reagents: Peptides from Stealth Peptides Inc. Powered by (Newton Centre, MA). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol, as described in Avanti Polar Lipids Inc. Purchased from (Alabaster, AL). Amplex Red peroxidase kit and Hepes buffer were purchased from Invitrogen (Carlsbad, CA). Cytochrome c and> 99.5% grade ethanol reagents were purchased from Sigma (St. Louis, MO).

Measurement of Cytochrome c Peroxidase Activity: Cytochrome c peroxidase activity was measured using the Amplex Red Peroxidase kit. Amplex Red reagent reacts with H ₂ O ₂ at a 1: 1 ratio in the presence of peroxidase to create a highly fluorescent compound called resorufin, which has an excitation at 570 nm and an emission at 585 nm. Cytochrome c at 2 μM was incubated in 20 mM Hepes buffer, pH 7.4, with free cholesterol or cholesterol-POPC liposomes (chol-POPC) dissolved in different concentrations of ethanol. 10 μM H ₂ O ₂ and 50 μM Amplex Red reagent were subsequently added. The fluorescence was then measured using time-lapse data collected from a Spectra Max 190 microplate spectrofluorimeter (Molecular Devices, Sunnyvale, CA).

  Preparation of Liposomes: Liposomes were prepared as described by Melzak et al. , Langmuir, 24: 9172-9180 (2008) as previously described. Chol-POPC liposomes were prepared at a molar ratio of 1: 1. The stock solutions were mixed and then allowed to dry under nitrogen for 30 minutes. After resuspension in 20 mM Hepes buffer, pH 7.4, the liposomes were extruded 25 times through a 50 nm pore membrane (Avestin).

  Cytochrome c reduction: 20 μM of cytochrome c in Hepes buffer, pH 7.4, was contacted with different concentrations of chol-POPC liposomes and peptides. After 5 minutes, 50 μM ascorbate (vitamin C) was added to promote reduction of the cytochrome c protein. The absorbance at 550 nm was then recorded using a spectrophotometer (Spectramax Gemini XPS, Molecular Devices, Sunnyvale, CA) to obtain kinetics after treatment with different concentrations of peptide and cholesterol. (See Basova et al., 2007).

  Circular dichroism spectroscopy: Circular dichroism (CD) spectra were acquired using an AVIA 62 DS spectrophotometer. Soret using CD10 mm path length for cells containing 20 mM Hepes (pH 7.4) and 10 mM cytochrome c in the presence and absence of 40 μM chol-POPC liposomes and / or 100 μM SS-31 CD spectra were analyzed between 375-450 nm to monitor the area.

  Absorption Spectrum: The effect of cholesterol and aromatic cationic peptide on the structure of cytochrome c was analyzed using absorption spectrum (Pinherio et al., 1997). Wild-type cytochrome c typically has a peak at around 410 nm (which represents the Soure region) which may have been affected by protein damage. Absorbance at 350-450 nm was analyzed using a Spectramax Gemini XPS spectrophotometer (Molecular Devices, Sunnyvale, CA).

Cholesterol aromatic cationic peptide - interaction with cytochrome c complex: D-Arg-2 ', 6'-Dmt-Lys-Ald-NH 2 ([ald] SS-31) is, D-Arg-2 'is a Aradan containing derivative of 6'-Dmt-Lys-Phe- NH 2 (SS-31) (aladan) (ald). Birk et al. , J Am Soc Nephrol. 24 (8): 1250-1261 (2013). As the polarity sensitive fluorescent amino acid Ald decreases its environmental polarity and its emission maximum (λ _max ) undergoes a blue shift, its emission intensity increases. Thus, D-Arg-2 ', interaction between 6'-Dmt-Lys-Ald- NH 2 ([ald] SS-31) and phospholipid causes an observable transition of the fluorescence spectrum of the peptide . Birk et al. , J Am Soc Nephrol. 24 (8): 1250-1261 (2013). D-Arg-2 of 390~600nm ', analyzes the fluorescence spectrum of 6'-Dmt-Lys-Ald- NH 2 ([ald] SS-31), peptide and cholesterol - interaction with cytochrome c complex Was evaluated.
Example 1 Cholesterol increases cytochrome c peroxidase activity in a dose dependent manner.

The Amplex Red Peroxidase kit was used to determine if cholesterol had an effect on the cytochrome c peroxidase activity. Cytochrome c was incubated with Amplex Red Reagent, hydrogen peroxide (H ₂ O ₂ ), and different concentrations of free cholesterol. As shown in FIG. 1, cholesterol increased cytochrome c peroxidase activity in a dose dependent manner. For example, incubation of cytochrome c with 300 μM free cholesterol resulted in a 40-fold increase in cytochrome c peroxidase activity. See FIG.

  Similar effects were also observed with chol-POPC liposomes. According to FIG. 1, incubation of cytochrome c with 300 μM chol-POPC liposomes resulted in a 48-fold increase in cytochrome c peroxidase activity. This indicates that cholesterol-induced upregulation of cytochrome c peroxidase activity is not specific for free cholesterol, but is also observed for cholesterol associated with lipid membranes.

These results indicate that cholesterol can impair mitochondrial function by increasing cytochrome c peroxidase activity.
Example 2 Aromatic cationic peptides inhibit cholesterol-induced cytochrome c peroxidase activity in a dose dependent manner.

Cytochrome c is incubated with D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) peptide or Phe-D-Arg-Phe-Lys-NH ₂ (SS-20) peptide And then treated with chol-POPC liposomes. As shown in FIG. 2, the aromatic-cationic peptide inhibited cholesterol-induced cytochrome c peroxidase activity in a dose dependent manner. Cytochrome c exposed to 300 μM chol-POPC liposomes and D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) compared to cytochrome c exposed only to chol-POPC liposomes Showed a 75% reduction in cytochrome c peroxidase activity. Similarly, cytochrome c exposed to 300 μM chol-POPC liposomes and Phe-D-Arg-Phe-Lys-NH ₂ (SS-20) compared to cytochrome c exposed to chol-POPC liposomes alone, as compared to cytochrome c. c showed a 77% decrease in peroxidase activity.

These results, D-Arg-2 ', aromatics such as 6'-Dmt-Lys-Phe- NH 2 (SS-31) or Phe-D-Arg-Phe- Lys-NH 2 (SS-20) FIG. 6 shows that cationic peptides can inhibit cholesterol-induced cytochrome c peroxidase activity. Thus, these results show that the aromatic-cationic peptides of the present technology are useful in methods for reducing cholesterol-induced cytochrome c peroxidase activity in subjects suffering from diseases characterized by cholesterol-induced mitochondrial dysfunction. Indicates that there is.
Example 3 Aromatic cationic peptides mitigate the effect of cholesterol on cytochrome c reduction.

  Cytochrome c must undergo reduction to function as an electron carrier. Cytochrome c reduction disorders contribute to mitochondrial dysfunction through the accumulation of ROS and lipid peroxides. Cytochrome c is unlikely to be reduced if it is damaged. (Basova et al., 2007).

Cytochrome c reduction was measured in the presence of 50 μM vitamin C (an antioxidant that decreases cytochrome c) with or without chol-POPC liposomes. FIG. 3A shows that cytochrome c exposed to cholesterol is less likely to be reduced as compared to cytochrome c not incubated with cholesterol. According to FIG. 3A, cholesterol inhibited cytochrome c reduction in a dose dependent manner. For example, incubation of cytochrome c with 300 μM chol-POPC liposomes resulted in a 21% reduction in cytochrome c reduction. See FIG. 3A. Cholesterol-induced impairment of cytochrome c reduction can be achieved by either D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) peptide or Phe-D-Arg-Phe-Lys-NH ₂ (SS- 20) Reversed by addition of any of the peptides. See Figure 3B.

These results, D-Arg-2 ', aromatics such as 6'-Dmt-Lys-Phe- NH 2 (SS-31) or Phe-D-Arg-Phe- Lys-NH 2 (SS-20) It is shown that cationic peptides can mitigate the effect of cholesterol on cytochrome c reduction. Thus, these results show that the aromatic-cationic peptide of the present technology is in a method for improving the inhibitory effect of cytochrome c reduction of cholesterol in a subject suffering from a disorder characterized by cholesterol-induced mitochondrial dysfunction Indicates that it is useful.
Example 4 D-Arg-2 ', 6' -Dmt-Lys-Phe-NH 2 (SS-31) peptides inhibit cholesterol induced structural changes of cytochrome c.

Circular dichroism (Olis DSM 20 spectropolarimeter), a probe for attachment of Met 80 residues to heme iron, was performed to analyze the Soret region (375-450 nm). Since the 416 nm dichroic band serves as a readout for the Met80-Fe (III) coordination in natural cytochrome c, the reduced intensity of the 416 nm Cotton effect (negative cotton peak) indicates a perturbed heme pocket region. (Santucci et al., 1997). As shown in FIG. 4, introduction of 40 μM chol-POPC liposome induced a structural change of cytochrome c. Specifically, the negative cotton peak at 416 nm is significantly reduced, demonstrating the absence of Met80-Fe (III) coordination. These data indicate that contact with cholesterol results in structural improvement of cytochrome c and exposure to heme iron. Since spectral disruption of the Fe-Met 80 coordination causes switching of the cytochrome c to the peroxidase from the electron carrier by increasing the exposure of heme Fe to H ₂ O ₂ , CD spectral data are shown in Examples 1 and 3 Match with the discovery of

FIG. 4 also shows the negative cotton peak of cytochrome c, which was treated with 100 μM D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) and incubated with chol-POPC liposomes. Indicates to recover. These results indicate that aromatic cationic peptides such as D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) restore cytochrome c structure and induce cholesterol in cholesterol c It shows that it has the ability to protect from structural damage.

The cholesterol-induced structural improvement of cytochrome c was also analyzed using absorption spectroscopy. Absorbance spectra at 350-450 nm were analyzed to monitor changes in the Soret band. The Soret band with maximum intensity at 410 nm indicates the presence of both axial ligands (His 18 and Met 80 bound to heme iron). (Pinherio et al., 1997). As shown in FIG. 5A, incubation with chol-POPC liposomes leads to a marked decrease in the intensity of the peak at 410 nm, which indicates damage or alteration to the structure of cytochrome c. FIG. 5B shows the intensity of the 410 nm peak of cytochrome c, which was treated with 100 μM D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) and incubated with chol-POPC liposomes. To increase the These results indicate that aromatic cationic peptides such as D-Arg-2 ', 6'-Dmt-Lys-Phe-NH ₂ (SS-31) restore cytochrome c structure and induce cholesterol in cholesterol c It shows that it has the ability to protect from structural damage. Thus, these results show that the aromatic-cationic peptide of the present technology is in a method for protecting cytochrome c from cholesterol-induced structural damage in a subject suffering from a disease characterized by cholesterol-induced mitochondrial dysfunction Indicates that it is useful.
Example 5 Interaction between aromatic-cationic peptide and cholesterol-cytochrome c complex.

Aromatic cationic peptide D-Arg-2 ', and 6'-Dmt-Lys-Ald- NH 2 ([ald] SS-31) Cholesterol - the interaction between cytochrome c complex, Birk et al. , And investigated using the emission spectrum technique described in 2013. As shown in FIG. 6, D-Arg-2 ', 6'-Dmt-Lys-Ald-NH ₂ ([ald] SS-31) exhibits a blue shift when encountering 100 μM cholesterol, which is That is, λ _max is shifted from 530 nm to 436 nm. D-Arg-2 ', 6'-Dmt-Lys-Ald-NH ₂ ([ald] SS-31) exhibits no migration when contacted with 4 μM cytochrome c, which means that λ _max is 530 nm It means that it stayed in These observations are described in Birk et al. , Consistent with the findings in 2013.

Birk et al. , 2013 is also smaller when D-Arg-2 ', 6'-Dmt-Lys-Ald-NH ₂ ([ald] SS-31) normally interacts with the lipid-cytochrome c complex It was shown to exhibit a blue shift. However, as shown in FIG. 6, D-Arg-2 ', 6'-Dmt-Lys-Ald-NH ₂ ([ald] SS-31), when contacted with both cholesterol and cytochrome c, It did not show a transition. These results, D-Arg-2 ', 6'-Dmt-Lys-Ald-NH 2 ([ald] SS-31) is effectively cholesterol - show that blocking the formation of cytochrome c complex .

Taken together, these results show that aromatic cationic peptides such as D-Arg-2 ', 6'-Dmt-Lys-Ald-NH ₂ ([ald] SS-31) are cholesterol-cytochrome c complexes. By inhibiting the formation it is shown to protect the structural integrity of cytochrome c against cholesterol induced damage. Thus, these results are useful in methods for the aromatic cationic peptides of the present technology to inhibit the formation of cholesterol-cytochrome c complex in a subject suffering from a disorder characterized by cholesterol-induced mitochondrial dysfunction To indicate that

Furthermore, these results show that the aromatic-cationic peptide of the present technology is useful in a method for inhibiting cholesterol-induced lipid peroxidation by disrupting the interaction between cytochrome c and cholesterol. Thus, it is shown to be useful in a method for reducing cardiolipin oxidation in a subject suffering from a disorder characterized by cholesterol induced mitochondrial dysfunction. Thus, these results indicate that the aromatic-cationic peptides of the present technology are useful in methods for treating a subject suffering from a disorder characterized by cholesterol-induced mitochondrial dysfunction.

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Equal

  The technology is not limited with respect to the particular embodiments described in the present application which are intended as single illustrations of individual aspects of the technology. Many modifications and variations of this technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and devices within the scope of the technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the appended claims. The technology is limited only by the terms of the appended claims, and the full scope of equivalents to which such claims are entitled. It should be understood that the technology is not limited to particular methods, reagents, compounds, compositions, or biological systems that may, of course, differ. Also, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  In addition, where features or aspects of the present disclosure are described in terms of Markush groups, those skilled in the art will recognize that the present disclosure is also described thereby with respect to any individual element or subset of elements of Markush groups Will.

  As will be appreciated by those skilled in the art, all ranges mentioned herein are all possible sub-ranges and sub-ranges thereof, particularly with regard to providing a written description for all purposes. Also includes combinations of Any range given is a simple statement that it is sufficient to fully explain and enable that the same range is at least equally divided into 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes etc. It can be done. As a non-limiting example, each range discussed herein may be easily divided into a lower third, a middle third, an upper third, and so on. Also, as will be appreciated by those skilled in the art, all terms such as "max", "at least", "super", "less than", etc., include the numbers cited and, as discussed above, subsequently subordinate It refers to a range that can be divided into ranges. Finally, as will be understood by those skilled in the art, the scope includes each individual element. Thus, for example, a group with 1-3 cells refers to a group with 1, 2 or 3 cells. Similarly, a group with 1-5 cells refers to a group with 1, 2, 3, 4 or 5 cells (hereinafter the same).

  All patents, patent applications, provisional applications, and publications mentioned or cited herein, to the extent that they do not conflict with the clear teachings herein, are referred to in their entirety including all figures and tables. Is incorporated by

  Other embodiments are within the scope of the following claims.

Claims (25)

A method for treating a subject suffering from a disorder characterized by cholesterol induced mitochondrial dysfunction, comprising
Said method comprising administering to said subject a therapeutically effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof. The aromatic cationic peptide, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys-NH 2 (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31), the method of claim 1.   The method according to claim 1, wherein the salt is acetate, tartrate, or trifluoroacetate. Said peptide, D-Arg-2 ', including 6'-Dmt-Lys-Phe- NH 2 (SS-31), The method of claim 1.   The method according to claim 1, wherein the disease is Niemann-Pick disease.   Treatment comprises alleviation or amelioration of one or more symptoms of Niemann-Pick disease, said symptoms of Niemann-Pick disease, difficulty moving the limbs (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly) Dementia, indistinct and irregular speech (arthrogenetic disorder), dysphoria (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, of moving eyes up and down Difficulty (vertical supranuclear gaze paralysis), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of cortical bone, distortion of hip bone, seizure, cherry red in the eye 6. The method of claim 5, which is one or more symptoms selected from the group consisting of spots, and sleep related disorders.   The method according to claim 1, wherein the aromatic cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation. A method for reducing cardiolipin oxidation in a subject suffering from a disorder characterized by cholesterol induced mitochondrial dysfunction, comprising
Administering the effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to the subject. The aromatic cationic peptide, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys-NH 2 (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31), the method of claim 8.   The method according to claim 8, wherein the salt is acetate, tartrate, or trifluoroacetate.   9. The method of claim 8, wherein the aromatic cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.   9. The method of claim 8, wherein the disease is Niemann-Pick disease.   The method comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, the symptoms comprising difficulty moving the limb (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly), dementia Indistinct, irregular speech (arthria), incongenital swallowing (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, difficulty moving eyes up and down (vertical) Supranuclear fixation), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of the cortical bone, distortion of the hipbone, seizures, cherry red spots in the eye, 13. The method of claim 12, wherein the disorder is a sleep related disorder. A method for reducing cholesterol induced cytochrome c peroxidase activity in a subject suffering from a disease characterized by cholesterol induced mitochondrial dysfunction, comprising
Administering the effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to the subject. The aromatic cationic peptide, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys-NH 2 (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31), the method of claim 14.   The method according to claim 14, wherein the salt is acetate, tartrate or trifluoroacetate.   15. The method of claim 14, wherein the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.   15. The method of claim 14, wherein the disease is Niemann-Pick disease.   The method comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, the symptoms comprising difficulty moving the limb (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly), dementia Indistinct, irregular speech (arthria), incongenital swallowing (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, difficulty moving eyes up and down (vertical) Supranuclear fixation), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of the cortical bone, distortion of the hipbone, seizures, cherry red spots in the eye, 19. The method of claim 18, wherein the disorder is a sleep related disorder. A method for protecting cytochrome c from cholesterol induced structural damage in a subject suffering from a disease characterized by cholesterol induced mitochondrial dysfunction, comprising
Administering the effective amount of an aromatic cationic peptide or a pharmaceutically acceptable salt thereof to the subject. Aromatic cationic peptide, 2 ', 6'-Dmt- D-Arg-Phe-Lys-NH 2 (SS-02), Phe-D-Arg-Phe-Lys-NH 2 (SS-20), and D-Arg-2 ', comprising one or more peptides selected from the group consisting of 6'-Dmt-Lys-Phe- NH 2 (SS-31), the method of claim 20.   21. The method of claim 20, wherein the salt is acetate, tartrate, or trifluoroacetate.   21. The method of claim 20, wherein the aromatic-cationic peptide is administered orally, parenterally, intravenously, subcutaneously, transdermally, topically, or by inhalation.   21. The method of claim 20, wherein the disease is Niemann-Pick disease.   The method comprises reducing or ameliorating one or more symptoms of Niemann-Pick disease, the symptoms comprising difficulty moving the limb (dystonia), hypertrophy of the spleen and liver (hepatosplenomegaly), dementia Indistinct, irregular speech (arthria), incongenital swallowing (dysphagia), sudden loss of muscle tone that can lead to falls (weakness seizures), tremor, difficulty moving eyes up and down (vertical) Supranuclear fixation), ataxia, loss of appetite, abdominal distension, thrombocytopenia, jaundice, pain, enlargement of the medullary cavity, thinning of the cortical bone, distortion of the hipbone, seizures, cherry red spots in the eye, 25. The method of claim 24, wherein the disorder is a sleep related disorder.